Compositions and methods for inhibiting group ii intron rna

ABSTRACT

The present invention provides compositions and methods for inhibiting group II intron splicing for treating or preventing a disease or disorder associated with an organism harboring an active group II intron. The present invention also provides compositions and methods for inhibiting group II intron splicing for inhibiting, preventing or reducing growth of an organism harboring an active group II intron.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of, and claims priority to, U.S.Application No. patent application Ser. No. 16/964,621 filed Jul. 24,2020, now allowed, which is a 35 U.S.C. § 371 national phase applicationfrom, and claims priority to, International Application No.PCT/US2019/015086, filed Jan. 25, 2019, and published under PCT Article21(2) in English, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/622,287, filed Jan. 26, 2018, all ofwhich applications are incorporated herein by reference in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under-AI115951 andGM050313 awarded by National Institutes of Health. The government hascertain rights in the invention.

SEQUENCE LISTING

This disclosure contains one or more sequences in a computer readableformat in an accompanying .xml file entitled “047162-7290US2_SeqListing.XML”, which is 25.3 KBytes in size and was created on Aug. 22,2023, the contents of which are incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION

It is becoming increasingly clear that large, highly structured RNAmolecules are essential for most metabolic functions. There has beencorresponding interest in developing small molecules that targetspecific RNA structures and thereby modulate gene expression for thetreatment of various diseases. Previous studies on bacterialriboswitches have demonstrated that folded RNA molecules can containhigh affinity pockets for the binding of small molecules such asmetabolites, and these ligands have been optimized in efforts to developnew antibiotics (Blount and Breaker, 2006, Nat Biotechnol,24:1558-1564). Indeed, antibacterial compounds have long been known totarget ribosomal RNA (Wallis and Schroeder, 1997, Prog Biophys Mol Biol,67:141-154; Schroeder et al., 2000, EMBO J, 19:1-9) and, givenavailability of high resolution structural data, these have been thesubject of continuing optimization (Wilson, 2009, Crit Rev Biochem MolBiol, 44:393-433; Wilson, 2014, Nature reviews. Microbiology, 12:35-48;Blaha et al., 2012, Current opinion in structural biology, 22:750-758).While these studies provide important precedents, they were conducted onRNA molecules that were already known to bind classes of smallmolecules. De-novo RNA targeting efforts have been limited, and theyhave focused primarily on small molecules that bind RNA secondarystructural elements, such as RNA hairpins from triplet repeat diseasesand stem-loops in viral RNA genomes (Bernat and Disney, 2015, Neuron,87:28-46; Disney, 2013, Drug discovery today, 18:1228-1236). Thus far,RNA tertiary structures have not been successfully targeted de-novousing high throughput screening and lead optimization techniques thatare typical of medicinal chemistry programs for the development ofprotein inhibitors. With the exception of bacterial riboswitchinhibitors, there are no new classes of antimicrobial compounds thathave been developed to target RNA, and none that are designed to targeteukaryotic pathogens.

Diseases involving pathogenic yeasts have become an increasing threat,particularly for recipients of implanted devices, for neonatal patients,cancer patients and others with compromised immune systems (Kett et al.,2011, Crit Care Med, 39:665-670). Specifically, there has been a markedincrease in pathologies associated with non-albican strains,particularly Candida parapsdosis (Guinea, 2014, Clin Microbiol Infect,20 Suppl 6:5-10). The most common treatment for critically ill patientswith candidaemia is Amphotericin B (AmpB), which is extremely toxic,causing numerous adverse effects. Azole derivatives and echinocandinshave been developed as alternatives to AmpB, but their use has beencomplicated by rapid resistance to Candida strains (Jensen et al., 2015,Antimicrobial agents and chemotherapy, 60:1500-1508; Arendrup andPerlin, 2014, Curr Opin Infect Dis, 27:484-492). The availability ofpotent antifungals that lack toxicity in mammals is therefore a majorunmet medical need, and of value for industrial and agriculturalapplications. Importantly, the development of new antifungals ischallenging because, as eukaryotes, fungi and yeast cells share enzymesand biochemical pathways that are structurally and functionally similarto those of humans. However, fungal RNA metabolism differssignificantly, thereby providing a potential route toward newtherapeutics.

Thus, there is a need in the art for compositions and methods fortargeting specific RNA structures for inhibiting and preventing thegrowth of organisms that harbor RNA capable of forming the targetstructure. The present invention satisfies this need.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a composition comprising aninhibitor of group II intron splicing.

In one embodiment, the inhibitor of group II intron splicing is at leastone selected from the group consisting of a protein, a peptide, apeptidomimetic, an antibody, a ribozyme, a small molecule chemicalcompound, a nucleic acid, an aptamer, a modified nucleic acid, a vector,a genome editing system and an antisense nucleic acid molecule.

In one embodiment, the inhibitor of group II intron splicing is a smallmolecule chemical compound.

In one embodiment, the inhibitor of group II intron splicing is a smallmolecule chemical compound of Formula (I) or a salt thereof;

wherein in Formula (I):

-   -   X is O, S, or NR¹⁰;    -   R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each        independently selected from the group consisting of H, —C₁-C₆        alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, aryl, heteroaryl,        cycloalkyl, heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂, —OH,        —OR¹¹, —SR¹¹, —S(═O)R¹¹, —S(═O)₂R¹¹, —NHS(═O)₂R¹¹, —C(═O)R¹¹,        —OC(═O)R¹¹, —CO₂R¹¹, —OCO₂R¹¹, —CH(R¹¹)₂, —N(R¹¹)₂,        —C(═O)N(R¹¹)₂, —C(═O)NHR¹¹, —OC(═O)N(R¹¹)₂, —NHC(═O)NH(R¹¹),        —NHC(═O)R¹¹, —NHC(═O)OR¹¹, —C(OH)(R¹¹)₂, and —C(NH₂)(R¹¹)₂, and        combinations thereof;    -   or optionally two adjacent R¹-R⁵ or R⁶-R⁹ are joined to form a        ring;    -   each occurrence of R¹¹ is independently selected from the group        consisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆        heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, F,        Cl, Br, I, —CN, —NO₂, —OH, and combinations thereof; and    -   wherein only one of the two following conditions is met:        -   condition 1, wherein the bond between carbons 1 and 2 is a            single bond, the bond between carbons 2 and 3 is a double            bond, Y is (═O), and Z is H; or        -   condition 2, wherein the bond between carbons 1 and 2 is a            double bond, the bond between carbons 2 and 3 is a single            bond, Y is H, and Z is (═O).

In one embodiment, the small molecule chemical compound of Formula (I)is a compound of Formula (II);

In one embodiment, the small molecule chemical compound of Formula (I)is a compound of Formula (III);

In one embodiment, R², R³, and R⁴ are OH. In one embodiment, R⁷ isrepresented by Formula (IV):

wherein in Formula (IV):

-   -   * represents the attachment to Formula (I);    -   A¹, A², A³, and A⁴ are CR¹³ or N;    -   R¹² is selected from the group consisting of H, C₁-C₆ alkyl,        C₁-C₆ fluoroalkyl, C₁-C₆ perfluoroalkyl, C₁-C₆ heteroalkyl,        aryl, heteroaryl, cycloalkyl, heterocycloalkyl, F, Cl, Br, I,        —CN, —NO₂, —OH, —OR¹⁴, —SR¹⁴, —S(═O)R¹⁴, —S(═O)₂R¹⁴,        —S(═O)₂NHR¹⁴, —S(═O)₂N(R¹⁴)₂, —NHS(═O)₂R¹⁴, —C(═O)R¹⁴,        —OC(═O)R¹⁴, —CO₂R¹⁴, —OCO₂R¹⁴, —CH(R¹⁴)₂, —N(R¹⁴)₂,        —C(═O)N(R¹⁴)₂, —C(═O)NHR¹⁴, —OC(═O)N(R¹⁴)₂, —NHC(═O)NH(R¹⁴),        —NHC(═O)R¹⁴, —NHC(═O)OR¹⁴, —C(OH)(R¹⁴)₂, and —C(NH₂)(R¹⁴)₂, and        combinations thereof;    -   each occurrence of R¹³ is independently selected from the group        consisting of H, C₁-C₆ alkyl, F, Cl, Br, I, —CN, —NO₂, —OH,        —CF₃, C₁-C₆ heteroalkyl, aryl-(C₁-C₃)alkyl, and cycloalkyl; or        optionally two adjacent R¹³ are joined to form a ring;    -   each occurrence of R¹⁴ is independently selected from the group        consisting of H, C₁-C₆ alkyl, F, Cl, Br, I, —CN, —NO₂, —OH,        —CF₃, C₁-C₆ heteroalkyl, aryl-(C₁-C₃)alkyl, and cycloalkyl;        or optionally two R¹⁴ on the same atom may together form a ring.

In one embodiment, R⁸ is —C(═O)NHR¹⁰.

In one embodiment, the compound of Formula (I) is selected one of:

In one embodiment, the inhibitor of group II intron splicing is a smallmolecule chemical compound of Formula (V) or a salt thereof;

wherein in Formula (V):

-   -   L is a divalent linking group selected from the group consisting        of a single bond and ethylene;    -   R²¹, R²², R²³, R²⁴, and R²⁵ are each independently selected from        the group consisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl,        —C₁-C₆ heteroalkyl, aryl, heteroaryl, cycloalkyl,        heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂, —OH, —OR²⁷, —SR²⁷,        —S(═O)R²⁷, —S(═O)₂R²⁷, —NHS(═O)₂R²⁷, —C(═O)R²⁷, —OC(═O)R²⁷,        —CO₂R²⁷, —OCO₂R²⁷, —CH(R²⁷)₂, —N(R²⁷)₂, —C(═O)N(R²⁷)₂,        —C(═O)NHR²⁷, —OC(═O)N(R²⁷)₂, —NHC(═O)NH(R²⁷), —NHC(═O)R²⁷,        —NHC(═O)OR²⁷, —C(OH)(R²⁷)₂, and —C(NH₂)(R²⁷)₂, and combinations        thereof;    -   or optionally two adjacent R²¹-R²⁵ are joined to form a ring;    -   each occurrence of R²⁷ is independently selected from the group        consisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆        heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, F,        Cl, Br, I, —CN, —NO₂, —OH, and combinations thereof; and    -   R²⁶ is selected from the group consisting of an aryl group and a        heteroaryl group, wherein the aryl or heteroaryl group may be        optionally substituted. In one embodiment, the R²², R²³, and R²⁴        are OH. In one embodiment, R²⁶ is a group of Formula (VI):

wherein in Formula (VI):

-   -   * represents the attachment to Formula (V);    -   Z¹, Z², Z³, Z⁴, and Z⁵ are CR²⁸ or N;    -   each occurrence of R²⁸ is independently selected from the group        consisting of H, C₁-C₆ alkyl, F, Cl, Br, I, —CN, —NO₂, —OH,        —CF₃, —OR²⁹, —N(R²⁹)₂, —C(═O)R²⁹, C₁-C₆ heteroalkyl,        aryl-(C₁-C₃)alkyl, cycloalkyl, alkynyl, and combinations        thereof; or optionally two adjacent R²⁸ are joined together to        form a ring; and    -   R²⁹ is selected from the group consisting of H, C₁-C₆ alkyl, and        heteroaryl-(C₁-C₃)alkyl.

In one embodiment, the compound of Formula (V) is one of:

In one embodiment, the invention relates to a method of reducing orpreventing growth of an organism harboring an active group II introncomprising contacting an organism harboring an active group II intronwith an effective amount of an inhibitor of group II intron splicing.

In one embodiment, the inhibitor of group II intron splicing is at leastone selected from the group consisting of a protein, a peptide, apeptidomimetic, an antibody, a ribozyme, a small molecule chemicalcompound, a nucleic acid, an aptamer, a modified nucleic acid, a vector,and an anti sense nucleic acid molecule.

In one embodiment, the inhibitor of group II intron splicing is a smallmolecule chemical compound.

In one embodiment, the inhibitor of group II intron splicing is a smallmolecule chemical compound of Formula (I) or a salt thereof;

wherein in Formula (I):

-   -   X is O, S, or NR¹⁰;    -   R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each        independently selected from the group consisting of H, —C₁-C₆        alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, aryl, heteroaryl,        cycloalkyl, heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂, —OH,        —OR¹¹, —SR¹¹, —S(═O)R¹¹, —S(═O)₂R¹¹, —NHS(═O)₂R¹¹, —C(═O)R¹¹,        —OC(═O)R¹¹, —CO₂R¹¹, —OCO₂R¹¹, —CH(R¹¹)₂, —N(R¹¹)₂,        —C(═O)N(R¹¹)₂, —C(═O)NHR¹¹, —OC(═O)N(R¹¹)₂, —NHC(═O)NH(R¹¹),        —NHC(═O)R¹¹, —NHC(═O)OR¹¹, —C(OH)(R¹¹)₂, and —C(NH₂)(R¹¹)₂, and        combinations thereof;    -   or optionally two adjacent R¹-R⁵ or R⁶-R⁹ are joined to form a        ring;    -   each occurrence of R¹¹ is independently selected from the group        consisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆        heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, F,        Cl, Br, I, —CN, —NO₂, —OH, and combinations thereof; and    -   wherein only one of the two following conditions is met:        -   condition 1, wherein the bond between carbons 1 and 2 is a            single bond, the bond between carbons 2 and 3 is a double            bond, Y is (═O), and Z is H; or        -   condition 2, wherein the bond between carbons 1 and 2 is a            double bond, the bond between carbons 2 and 3 is a single            bond, Y is H, and Z is (═O).

In one embodiment, the inhibitor of group II intron splicing is a smallmolecule chemical compound of Formula (V) or a salt thereof;

wherein in Formula (V):

-   -   L is a divalent linking group selected from the group consisting        of a single bond and ethylene;    -   R²¹, R²², R²³, R²⁴, and R²⁵ are each independently selected from        the group consisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl,        —C₁-C₆ heteroalkyl, aryl, heteroaryl, cycloalkyl,        heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂, —OH, —SR²⁷,        —S(═O)R²⁷, —S(═O)₂R²⁷, —NHS(═O)₂R²⁷, —C(═O)R²⁷, —OC(═O)R²⁷,        —CO₂R²⁷, —OCO₂R²⁷, —CH(R²⁷)₂, —N(R²⁷)₂, —C(═O)N(R²⁷)₂,        —C(═O)NHR²⁷, —OC(═O)N(R²⁷)₂, —NHC(═O)NH(R²⁷), —NHC(═O)R²⁷,        —NHC(═O)OR²⁷, —C(OH)(R²⁷)₂, and —C(NH₂)(R²⁷)₂, and combinations        thereof;    -   or optionally two adjacent R²¹-R²⁵ are joined to form a ring;    -   each occurrence of R²⁷ is independently selected from the group        consisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆        heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, F,        Cl, Br, I, —CN, —NO₂, —OH, and combinations thereof; and    -   R²⁶ is selected from the group consisting of an aryl group and a        heteroaryl group, wherein the aryl or heteroaryl group may be        optionally substituted.

In one embodiment, the invention relates to a method of inhibiting,treating or preventing a disease associated with an organism harboringan active group II intron in a subject comprising administering aneffective amount of a composition comprising an inhibitor of group IIintron splicing to a subject in need thereof. In one embodiment, thedisease or disorder is selected from the group consisting of a bacterialinfection, a yeast infection, a fungal infection, and a parasiteinfection. In one embodiment, the subject is a mammal. In oneembodiment, the mammal is a human. In one embodiment, the inhibitor ofgroup II intron splicing is at least one of a protein, a peptide, apeptidomimetic, an antibody, a ribozyme, a small molecule chemicalcompound, a nucleic acid, an aptamer, a modified nucleic acid, a vector,and an antisense nucleic acid molecule.

In one embodiment, the inhibitor of group II intron splicing is a smallmolecule chemical compound.

In one embodiment, the inhibitor of group II intron splicing is a smallmolecule chemical compound of Formula (I) or a salt thereof;

wherein in Formula (I):

-   -   X is O, S, or NR¹⁰;    -   R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each        independently selected from the group consisting of H, —C₁-C₆        alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, aryl, heteroaryl,        cycloalkyl, heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂, —OH,        —OR¹¹, —SR¹¹, —S(═O)R¹¹, —S(═O)₂R¹¹, —NHS(═O)₂R¹¹, —C(═O)R¹¹,        —OC(═O)R¹¹, —CO₂R¹¹, —OCO₂R¹¹, —CH(R¹¹)₂, —N(R¹¹)₂,        —C(═O)N(R¹¹)₂, —C(═O)NHR¹¹, —OC(═O)N(R¹¹)₂, —NHC(═O)NH(R¹¹),        —NHC(═O)R¹¹, —NHC(═O)OR¹¹, —C(OH)(R¹¹)₂, and —C(NH₂)(R¹¹)₂, and        combinations thereof;    -   or optionally two adjacent R¹-R⁵ or R⁶-R⁹ are joined to form a        ring;    -   each occurrence of R¹¹ is independently selected from the group        consisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆        heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, F,        Cl, Br, I, —CN, —NO₂, —OH, and combinations thereof; and    -   wherein only one of the two following conditions is met:        -   condition 1, wherein the bond between carbons 1 and 2 is a            single bond, the bond between carbons 2 and 3 is a double            bond, Y is (═O), and Z is H; or        -   condition 2, wherein the bond between carbons 1 and 2 is a            double bond, the bond between carbons 2 and 3 is a single            bond, Y is H, and Z is (═O).

In one embodiment, the inhibitor of group II intron splicing is a smallmolecule chemical compound of Formula (V) or a salt thereof;

wherein in Formula (V):

-   -   L is a divalent linking group selected from the group consisting        of a single bond and ethylene;    -   R²¹, R²², R²³, R²⁴, and R²⁵ are each independently selected from        the group consisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl,        —C₁-C₆ heteroalkyl, aryl, heteroaryl, cycloalkyl,        heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂, —OH, —SR²⁷,        —S(═O)R²⁷, —S(═O)₂R²⁷, —NHS(═O)₂R²⁷, —C(═O)R²⁷, —OC(═O)R²⁷,        —CO₂R²⁷, —OCO₂R²⁷, —CH(R²⁷)₂, —N(R²⁷)₂, —C(═O)N(R²⁷)₂,        —C(═O)NHR²⁷, —OC(═O)N(R²⁷)₂, —NHC(═O)NH(R²⁷), —NHC(═O)R²⁷,        —NHC(═O)OR²⁷, —C(OH)(R²⁷)₂, and —C(NH₂)(R²⁷)₂, and combinations        thereof;    -   or optionally two adjacent R²¹-R²⁵ are joined to form a ring;    -   each occurrence of R²⁷ is independently selected from the group        consisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆        heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, F,        Cl, Br, I, —CN, —NO₂, —OH, and combinations thereof; and    -   R²⁶ is selected from the group consisting of an aryl group and a        heteroaryl group, wherein the aryl or heteroaryl group may be        optionally substituted.

In one embodiment, the organism is selected from the group consisting ofa bacteria, a yeast, a fungus, a protist, a parasite, and a plant.

In one embodiment, administration of the inhibitor reduces or preventsat least one of biofilm and algae formation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict a fluorescent assay developed to screen inhibitors ofgroup II intron splicing. FIG. 1A depicts the sequences of exemplaryfluorescently labeled hydrolysable (SEQ ID NO:9) and non-hydrolyzable(SEQ ID NO:10) substrates used in this study. FIG. 1B depicts aschematic of the fluorescent assay used for high throughput screening(HTS) and for determination of the IC₅₀ values shown in Table 2. FIG. 1Cdepicts exemplary experimental results demonstrating that fluorescenceis only observed when the ribozyme is incubated with the substrate underproper reaction conditions (in the presence of magnesium ions). FIG. 1Ddepicts examples of hits identified by HTS.

FIG. 2 depicts a chemical reaction diagram of the synthesis of5-Bromobenzofuran-3-yl)(3,4,5-tris(benzyloxy)phenyl)methanone.

FIG. 3 depicts a chemical reaction diagram of the synthesis ofBenzofuran-2-yl(3,4,5-trihydroxyphenyl)methanone (APY-068).

FIG. 4 depicts a chemical reaction diagram of the synthesis of5-[(5,7-Dichloro-1-benzofuran-2-yl)carbonyl]benzene-1,2,3-triol(APY-083).

FIG. 5 depicts a chemical reaction diagram of the synthesis of5-{[5-(4-Methylpyridin-3-yl)-1-benzofuran-2-yl]carbonyl}benzene-1,2,3-triol(APY-081).

FIG. 6 depicts a chemical reaction diagram of the synthesis of5-({5-[4(Hydroxymethyl)phenyl]-1-benzofuran-2-yl}carbonyl)benzene-1,2,3-triol(APY-084).

FIG. 7 depicts a chemical reaction diagram of the synthesis of5-[(5-{4-[(Diethylamino)methyl]phenyl}-1-benzofuran-2-yl)carbonyl]benzene-1,2,3-triolhydrobromide (APY-090).

FIG. 8 depicts a chemical reaction diagram of the synthesis ofN,N-Diethyl-4-{2-[(3,4,5-trihydroxyphenyl)carbonyl]-1-benzofuran-5-yl}benzamide(APY-091).

FIG. 9 depicts a chemical reaction diagram of the synthesis ofN-Butyl-4-{2-[(3,4,5-trihydroxyphenyl)carbonyl]-1-benzofuran-5-yl}benzene-1-sulfonamide(APY-094).

FIG. 10 depicts a chemical reaction diagram of the synthesis of1-(4-{2-[(3,4,5-Trihydroxyphenyl)carbonyl]-1-benzofuran-5-yl}phenyl)ethan-1-one(APY-101).

FIG. 11 depicts a chemical reaction diagram of the synthesis of5-({5-[6-(Trifluoromethyl)pyridin-3-yl]-1-benzofuran-2-yl}carbonyl)benzene-1,2,3-triol(APY-097).

FIG. 12 depicts a chemical reaction diagram of the synthesis of(Z)-2-(3,4,5-Trihydroxybenzylidene)benzofuran-3(2H)-one (APY-024).

FIG. 13 depicts a chemical reaction diagram of the synthesis of(Z)-2-(3,4-Dimethoxybenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one(APY-001).

FIG. 14 depicts a chemical reaction diagram of the synthesis of(Z)-2-(3,4-Dihydroxybenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one(APY-007).

FIG. 15 depicts a chemical reaction diagram of the synthesis of(Z)-2-(3,5-Dihydroxybenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one(APY-014).

FIG. 16 depicts a chemical reaction diagram of the synthesis of(Z)-4-Hydroxy-2-(3,4,5-trihydroxybenzylidene)benzofuran-3(2H)-one(APY-019).

FIG. 17 depicts a chemical reaction diagram of the synthesis ofN-[(3,4-Dimethoxyphenyl)methyl]-3,4,5-trihydroxybenzamide (APY-077).

FIG. 18 depicts a chemical reaction diagram of the synthesis of2-(4-Bromophenyl)-4-[3,4,5-tris(benzyloxy)phenyl]-1,3-thiazole(Intermediate 2).

FIG. 19 depicts a chemical reaction diagram of the synthesis ofN-Butyl-4-{4-[4-(3,4,5-trihydroxyphenyl)-1,3-thiazol-2-yl]phenyl}benzene-1-sulfonamide(APY-093).

FIG. 20 depicts a chemical reaction diagram of the synthesis of[3-Fluoro-4-({5-[4-(hydroxymethyl)phenyl]-1-benzofuran-2-yl}carbonyl)phenyl)boronicacid (APY-098).

FIG. 21 depicts a chemical reaction diagram of the synthesis ofN-(2-(pyrrolidin-1-yl)ethyl)-2-(3,4,5-trihydroxybenzoyl)benzofuran-5-carboxamide(NED-2020).

FIGS. 22A-22C depict the RNA target and assays for compound development.FIG. 22A depicts a crystal structure of the Oceaonobacillus iheyensis(O.i.) group II intron. The active site domain of the ribozyme is shownin black, and the scaffolding domains in gray. Cavities in the molecule(identified by the PyMol Molecular Graphics System, version 1.8Schroedinger, LLC) shown as potential sites for binding of smallmolecules, are indicated with space filling. FIG. 22B (top), depicts aschematic of the fluorescent assay used for HTS and for the IC₅₀determination, and (bottom) representative IC₅₀ values (Table 2) wereobtained by plotting relative fluorescence vs the inhibitorconcentration. FIG. 22C (top), depicts the schematic of theself-splicing assay used to determine K_(i) values for the group IIintron splicing inhibitors, and (bottom) representative K_(i) values(FIG. 24 , Table 2) were determined by plotting the rate constants(k_(obs) values) versus inhibitor concentration.

FIGS. 23A-23B depict an exemplary analysis of the secondary structure ofa representative yeast intron, in this case the ai5γ group II intron,which is located within the COX1 gene of Saccharomyces cerevisiae(S.c.). FIG. 23A depicts a diagram of the secondary structure of theai5γ S. cerevisiae group II intron with short exons. Long-range tertiarycontacts are indicated by Greek letters. FIG. 23B depicts an alignmentof the catalytic Domain 5 from the ai5γ group II intron from S.cerevisiae (SEQ ID NO:11) and from the group II intron from Candidaparapsilosis (SEQ ID NO:12).

FIG. 24 depicts a summary of structure-activity relationships (SAR)studies on Compound 1. In vitro splicing inhibition constants (K_(i))for the S.c. ai5γ intron, MIC values for C. parapsilosis group II intronand IC₅₀ values for cytotoxicity in HEK-293 cells are shown next to eachmolecule. Changes that were introduced for optimization of inhibitoryactivity are indicated with an asterisk (*).

FIGS. 25A-25D depict exemplary experimental results demonstrating thedetermination of K_(i) values for compounds of interest. FIG. 25Adepicts representative gels showing lariat intron formation in theabsence (top) and in the presence of indicated concentrations ofAPY-024. FIG. 25A depicts representative time courses of lariat intronformation in the presence and in the absence of APY-024 at indicatedconcentrations ranging from 5 nM to 1 mM. Fraction of lariat intron wasquantified at each time point and plotted vs time to determine k_(obs)for each concentration of the inhibitor. These values were then plottedvs the inhibitor concentration to determine the Ki values for eachinhibitor (Table 1). FIG. 25C depicts representative plots of k_(obs)versus the inhibitor concentrations for different inhibitors, which werethen used to determine the Ki values (Table 1). FIG. 25D depictsrepresentative time courses of lariat intron formation before and afterten-fold dilution of inhibitor (Intronistat B (NED-2020)). Allexperiments were repeated twice to ensure reproducibility. Errorbars=s.e.m.

FIGS. 26A-26B depict exemplary experimental results demonstrating thatactive compounds selectively inhibit group II splicing in vivo. FIG. 26Adepicts a comparison of wild-type (WT) and intronless S. cerevisiaestrain growth in the presence of APY-101 (Intronistat A). From left toright, approximately 22,500, 4,500, and 900 cells of the indicatedstrains were plated on either YP 2% glucose or YP 3% glycerol 3% ethanolmedia containing DMSO vehicle or APY-101 (Intronistat A) at 128 μg/mLfor growth at 30° C. FIG. 26B depicts experimental results demonstratingthat active compounds inhibit splicing of COX1 aI5 g group II intron invivo. S. cerevisiae were grown in the presence of DMSO vehicle, activecompounds NED-2020 (Intronistat B), APY-101 (Intronistat A), andAPY-081, inactive compound APY-014, or amphotericin B (AmphB). Relativelevels of total and unspliced levels of COX1 indicated by qRT-PCRquantification of amplicons covering the eighth exon (total) or theintron-exon junction from the group IIA introns all and aI2, the group Iintrons aI5α and aI5β, or the group IIB intron aI5γ. Levels of total andunspliced nuclear-encoded YRA1 and MTR2 indicated by quantification ofamplicons covering an exon (total) or intron-exon junction of thesplicesomal targeted intron. Mean values and s.e.m. from n=4 independentexperiments are shown with ACT1 as a standard.

FIGS. 27A-27B depict exemplary experimental results demonstrating thatsmall molecules that are active in vitro cause a severe in vivo splicingdefect as evident from significant accumulation of precursor RNAmolecules containing 5′-exon-intron junction. FIG. 27A depicts acomparison of wild-type (WT) and intronless S. cerevisiae strain growthin the presence of active compound Intronistat A. From left to right,approximately 22,500, 4,500, and 900 cells of the indicated strains wereplated on YP 3% glycerol 3% ethanol media containing Intronistat A at 64μg/mL for growth at 30° C. FIG. 27B depicts that active compoundsinhibit splicing of COX1 aI5γ group II intron in vivo. S. cerevisiaewere grown in the presence of DMSO vehicle, active compound NED-2020(Intronistat B), APY-101 (Intronistat A), and APY-081, inactive APY-014,or amphotericin B (AmphB). Relative levels of total and unspliced levelsof COX1 indicated by qRT-PCR quantification of amplicons covering theeighth exon (total) or the intron-exon junction from the group IIAintrons all and aI2, the group I introns aI5α and aI5β, or the group IIBintron aI5γ. Levels of total and unspliced nuclear-encoded YRA1 and MTR2indicated by quantification of amplicons covering an exon (total) orintron-exon junction of the splicesomal targeted intron. Mean values ands.e.m. from n=4 independent experiments are shown with PGK1 as astandard. FIG. 27C depicts that C. parapsilosis COX1 exhibits a mildsplicing defect in the presence of active compound. C. parapsilosis weregrown in the presence of DMSO vehicle, active compound Intronistat B, orinactive compound APY-001. Relative levels of total and unspliced levelsof C.p.COX1 indicated by qRT-PCR quantification of amplicons coveringthe exon (total) or the intron-exon junction from the group JIB intron.Mean values and s.e.m. from n=3 independent experiments are shown withPGK1 as a standard.

FIGS. 28A-28B depict exemplary experimental results demonstrating thatactive compounds selectively inhibit group II splicing in vitro. FIG.28A depicts the inhibition of the ai5γ group II intron splicing in thepresence of excess of different RNAs: unlabeled SE RNA, intronic domainsD1, D3 and D56, yeast U2-U6 hairpin and yeast tRNA_(Phe). The averageand s.e.m. (error bars) are calculated from n=3 independent experiments.FIG. 28B depicts group I intron splicing is unaffected by the mostpotent inhibitor of group IIB intron splicing, Intronistat B.Representative time courses of Azoarcus pre-tRNA (Ile) group I intronsplicing in the absence and in the presence of the inhibitor IntronistatB at indicated concentrations. Fraction of precursor was quantified ateach time point, plotted vs time and fit to a double-exponentialequation to determine the reaction rate constants (1 min⁻¹ for the fastpopulation and 0.02 min⁻¹ for the slow population). Data representaverage of n=3 independent experiments, error bars are s.e.m.

FIGS. 29A-29B exemplary experimental results demonstrating that activecompound Intronistat B demonstrates minimal cytotoxicity. FIG. 29Adepicts that HEK-293T cells were grown in the presence of compounds atconcentrations ranging from 0.125 μg/ml to 128 μg/ml (0.031 μg/ml to 32μg/ml for Terfenadine, which was used as a cytotoxic control). After 24hours, viability was assessed with the luminescent Cell Titer Glo cellviability assay and IC₅₀ values (Table 2) were determined by fitting thedata to a 4-parameter logistic function. FIG. 29B depicts cells weregrown as in (FIG. 29A), but analyzed for viability after 72 hours growthin the presence of compounds as described in Methods. IC₅₀ values forcompounds APY-081, APY-084 and Intronistat A after 72-hour incubationwere 0.79±0.09 μg/ml, 1.00±0.04 μg/ml and 1.3±0.1 μg/ml, respectively.Data represent average of n=4 independent experiments for APY-081,Intronistat A and Intronisat B, and n=3 independent experiments forAPY-084. Error bars=s.e.m.

DETAILED DESCRIPTION

The present invention relates to compositions and methods for inhibitinggroup II intron splicing. In some embodiments, the invention relates tocompositions and methods for inhibiting group II intron splicing for useas anti-microbial and anti-fungal agents. The invention is based, inpart, on the unexpected discovery that group II introns fold into astructure that can be targeted to prevent the intron from splicing.Moreover, small molecule inhibitors of group II intron splicing havebeen identified as potential therapeutic compounds that can preventgrowth of organisms that harbor group II introns.

In one embodiment, the composition of the invention comprises aninhibitor of group II intron splicing. In one embodiment, the inhibitorof group II intron splicing is any compound, molecule, or agent thatreduces, inhibits, or prevents the self-splicing or ribozyme function ofa group II intron.

In one embodiment, the method of the present invention comprisesinhibiting group II intron splicing. In one embodiment, the inventionprovides a method for preventing, inhibiting, or disrupting the growthof organisms that harbor group II introns.

In one embodiment, the method is useful for treating or preventing adisease associated with an organism that harbors a group II intron. Inone embodiment, the method comprises administering to a subject aneffective amount of a composition comprising an inhibitor of group IIintron splicing.

In one embodiment, the method is useful for inhibiting or disruptinggrowth of organisms that harbor group II introns. For example, themethod can be used to inhibit or disrupt growth of organisms that harborgroup II introns in a commercial, industrial, or marine setting. Forexample, in certain embodiments, the method can be used to inhibit ordisrupt growth of organisms that harbor group II introns on a kitchen,bathroom, or pool surface. In certain embodiments, the method can beused to inhibit or disrupt growth of organisms that harbor group IIintrons on marine surfaces, including, but not limited to, piers, docks,boats, buoys, and the like.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention.

Generally, the nomenclature used herein and the laboratory procedures incell culture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization are those well-known and commonly employedin the art.

Standard techniques are used for nucleic acid and peptide synthesis. Thetechniques and procedures are generally performed according toconventional methods in the art and various general references (e.g.,Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach,Cold Spring Harbor Press, Cold Spring Harbor, NY, and Ausubel et al.,2002, Current Protocols in Molecular Biology, John Wiley & Sons, NY),which are provided throughout this document.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value,as such variations are appropriate to perform the disclosed methods.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics which arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

“Antisense” refers particularly to the nucleic acid sequence of thenon-coding strand of a double stranded DNA molecule encoding a protein,or to a sequence which is substantially homologous to the non-codingstrand. As defined herein, an antisense sequence is complementary to thesequence of a double stranded DNA molecule encoding a protein. It is notnecessary that the antisense sequence be complementary solely to thecoding portion of the coding strand of the DNA molecule. The antisensesequence may be complementary to regulatory sequences specified on thecoding strand of a DNA molecule encoding a protein, which regulatorysequences control expression of the coding sequences.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

A disease or disorder is “alleviated” if the severity of a sign orsymptom of the disease or disorder, the frequency with which such a signor symptom is experienced by a patient, or both, is reduced.

An “effective amount” or “therapeutically effective amount” of acompound is that amount of a compound which is sufficient to provide abeneficial effect to the subject to which the compound is administered.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of a compound, composition, vector,or delivery system of the invention in the kit for effecting alleviationof the various diseases or disorders recited herein. Optionally, oralternately, the instructional material can describe one or more methodsof alleviating the diseases or disorders in a cell or a tissue of amammal. The instructional material of the kit of the invention can, forexample, be affixed to a container which contains the identifiedcompound, composition, vector, or delivery system of the invention or beshipped together with a container which contains the identifiedcompound, composition, vector, or delivery system. Alternatively, theinstructional material can be shipped separately from the container withthe intention that the instructional material and the compound be usedcooperatively by the recipient.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in vivo, amenable to the methods described herein.In some non-limiting embodiments, the patient, subject or individual isa human.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs or symptoms of a disease or disorder, for the purpose ofdiminishing or eliminating those signs or symptoms.

As used herein, “treating a disease or disorder” means reducing theseverity and/or frequency with which a sign or symptom of the disease ordisorder is experienced by a patient.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific.

In some instances, the terms “specific binding” or “specificallybinding,” can be used in reference to the interaction of an antibody, aprotein, a peptide, or a nucleic acid (e.g., RNA or DNA) with a secondchemical species, to mean that the interaction is dependent upon thepresence of a particular sequence or structure (e.g., a specificnucleotide sequence or epitope) on the chemical species; for example, ansiRNA or riboswitch recognizes and binds to a specific nucleotidesequence rather than to nucleic acid molecules generally. If a siRNA isspecific for sequence “A”, the siRNA will specifically bind to amolecule containing sequence A.

A “coding region” of a gene consists of the nucleotide residues of thecoding strand of the gene and the nucleotides of the non-coding strandof the gene which are homologous with or complementary to, respectively,the coding region of an mRNA molecule which is produced by transcriptionof the gene.

A “coding region” of a mRNA molecule also consists of the nucleotideresidues of the mRNA molecule which are matched with an anti-codonregion of a transfer RNA molecule during translation of the mRNAmolecule or which encode a stop codon. The coding region may thusinclude nucleotide residues comprising codons for amino acid residueswhich are not present in the mature protein encoded by the mRNA molecule(e.g., amino acid residues in a protein export signal sequence).

“Complementary” as used herein to refer to a nucleic acid, refers to thebroad concept of sequence complementarity between regions of two nucleicacid strands or between two regions of the same nucleic acid strand. Itis known that an adenine residue of a first nucleic acid region iscapable of forming specific hydrogen bonds (“base pairing”) with aresidue of a second nucleic acid region which is antiparallel to thefirst region if the residue is thymine or uracil. Similarly, it is knownthat a cytosine residue of a first nucleic acid strand is capable ofbase pairing with a residue of a second nucleic acid strand which isantiparallel to the first strand if the residue is guanine. A firstregion of a nucleic acid is complementary to a second region of the sameor a different nucleic acid if, when the two regions are arranged in anantiparallel fashion, at least one nucleotide residue of the firstregion is capable of base pairing with a residue of the second region.In one embodiment, the first region comprises a first portion and thesecond region comprises a second portion, whereby, when the first andsecond portions are arranged in an antiparallel fashion, at least about50%, at least about 75%, at least about 90%, or at least about 95% ofthe nucleotide residues of the first portion are capable of base pairingwith nucleotide residues in the second portion. In one embodiment, allnucleotide residues of the first portion are capable of base pairingwith nucleotide residues in the second portion.

The term “DNA” as used herein is defined as deoxyribonucleic acid.

The term “RNA” as used herein is defined as ribonucleic acid.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

The term “expression vector” as used herein refers to a vectorcontaining a nucleic acid sequence coding for at least part of a geneproduct capable of being transcribed. In some cases, RNA molecules arethen translated into a protein, polypeptide, or peptide. In other cases,these sequences are not translated, for example, in the production ofantisense molecules, siRNA, ribozymes, and the like. Expression vectorscan contain a variety of control sequences, which refer to nucleic acidsequences necessary for the transcription and possibly translation of anoperatively linked coding sequence in a particular host organism. Inaddition to control sequences that govern transcription and translation,vectors and expression vectors may contain nucleic acid sequences thatserve other functions as well.

The term “fusion polypeptide” refers to a chimeric protein containing aprotein of interest (e.g., luciferase) joined to a heterologous sequence(e.g., a non-luciferase amino acid or protein).

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). Homology isoften measured using sequence analysis software (e.g., Sequence AnalysisSoftware Package of the Genetics Computer Group. University of WisconsinBiotechnology Center. 1710 University Avenue. Madison, Wis. 53705). Suchsoftware matches similar sequences by assigning degrees of homology tovarious substitutions, deletions, insertions, and other modifications.Conservative substitutions typically include substitutions within thefollowing groups: glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in its normal context in aliving animal is not “isolated,” but the same nucleic acid or peptidepartially or completely separated from the coexisting materials of itsnatural context is “isolated.” An isolated nucleic acid or protein canexist in substantially purified form, or can exist in a non-nativeenvironment such as, for example, a host cell.

The term “isolated” when used in relation to a nucleic acid, as in“isolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant with which it is ordinarily associated in its source. Thus,an isolated nucleic acid is present in a form or setting that isdifferent from that in which it is found in nature. In contrast,non-isolated nucleic acids (e.g., DNA and RNA) are found in the statethey exist in nature. For example, a given DNA sequence (e.g., a gene)is found on the host cell chromosome in proximity to neighboring genes;RNA sequences (e.g., a specific mRNA sequence encoding a specificprotein), are found in the cell as a mixture with numerous other mRNAsthat encode a multitude of proteins. However, isolated nucleic acidincludes, by way of example, such nucleic acid in cells ordinarilyexpressing that nucleic acid where the nucleic acid is in a chromosomallocation different from that of natural cells, or is otherwise flankedby a different nucleic acid sequence than that found in nature. Theisolated nucleic acid or oligonucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acidor oligonucleotide is to be utilized to express a protein, theoligonucleotide contains at a minimum, the sense or coding strand (i.e.,the oligonucleotide may be single-stranded), but may contain both thesense and anti-sense strands (i.e., the oligonucleotide may bedouble-stranded).

The term “isolated” when used in relation to a polypeptide, as in“isolated protein” or “isolated polypeptide” refers to a polypeptidethat is identified and separated from at least one contaminant withwhich it is ordinarily associated in its source. Thus, an isolatedpolypeptide is present in a form or setting that is different from thatin which it is found in nature. In contrast, non-isolated polypeptides(e.g., proteins and enzymes) are found in the state they exist innature.

By “nucleic acid” is meant any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, phosphorothioate, methylphosphonate, phosphorodithioate,bridged phosphorothioate or sulfone linkages, and combinations of suchlinkages. The term nucleic acid also specifically includes nucleic acidscomposed of bases other than the five biologically occurring bases(adenine, guanine, thymine, cytosine and uracil). The term “nucleicacid” typically refers to large polynucleotides.

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences”; sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

By “expression cassette” is meant a nucleic acid molecule comprising acoding sequence operably linked to promoter/regulatory sequencesnecessary for transcription and, optionally, translation of the codingsequence.

The term “operably linked” as used herein refers to the linkage ofnucleic acid sequences in such a manner that a nucleic acid moleculecapable of directing the transcription of a given gene and/or thesynthesis of a desired protein molecule is produced. The term alsorefers to the linkage of sequences encoding amino acids in such a mannerthat a functional (e.g., enzymatically active, capable of binding to abinding partner, capable of inhibiting, etc.) protein or polypeptide isproduced.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulator sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in an inducible manner.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced substantially only when aninducer which corresponds to the promoter is present.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR, and thelike, and by synthetic means.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

As used herein, a “peptidomimetic” is a compound containing non-peptidicstructural elements that is capable of mimicking the biological actionof a parent peptide. A peptidomimetic may or may not comprise peptidebonds.

The term “RNA” as used herein is defined as ribonucleic acid.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell.

A recombinant polynucleotide may serve a non-coding function (e.g.,promoter, origin of replication, ribosome-binding site, etc.) as well.

The term “recombinant polypeptide” as used herein is defined as apolypeptide produced by using recombinant DNA methods.

As used herein, “conjugated” refers to covalent attachment of onemolecule to a second molecule.

As used herein, the term “transdominant negative mutant” refers to anucleic acid encoding a polypeptide or protein product that preventsother copies of the same gene or gene product, which have not beenmutated (i.e., which have the wild-type sequence) from functioningproperly (e.g., by inhibiting wild type protein function). The productof a transdominant negative mutant nucleic acid is referred to herein as“dominant negative” or “DN” (e.g., a dominant negative protein, or a DNprotein).

The phrase “inhibit,” as used herein, means to reduce a molecule, areaction, an interaction, a gene, an mRNA, and/or a protein'sexpression, stability, function or activity by a measurable amount or toprevent entirely. Inhibitors are compounds that, e.g., bind to,partially or totally block stimulation, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate a protein, a gene,and an mRNA stability, expression, function and activity, e.g.,antagonists.

The phrase “anti-bacterial agent,” “antibiotic” as used herein means acompound or composition useful in reducing or preventing the viabilityor proliferation of a bacteria; or in treating or preventing a bacterialinfection.

The phrase “anti-fungal agent,” as used herein means a compound orcomposition useful in reducing or preventing the viability orproliferation of a fungus or yeast; or in treating or preventing afungal infection.

“Variant” as the term is used herein, is a nucleic acid sequence or apeptide sequence that differs in sequence from a reference nucleic acidsequence or peptide sequence respectively, but retains essentialbiological properties of the reference molecule. Changes in the sequenceof a nucleic acid variant may not alter the amino acid sequence of apeptide encoded by the reference nucleic acid, or may result in aminoacid substitutions, additions, deletions, fusions and truncations.Changes in the sequence of peptide variants are typically limited orconservative, so that the sequences of the reference peptide and thevariant are closely similar overall and, in many regions, identical. Avariant and reference peptide can differ in amino acid sequence by oneor more substitutions, additions, deletions in any combination. Avariant of a nucleic acid or peptide can be a naturally occurring suchas an allelic variant, or can be a variant that is not known to occurnaturally. Non-naturally occurring variants of nucleic acids andpeptides may be made by mutagenesis techniques or by direct synthesis.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

As used herein, the term “alkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a straight or branched chainhydrocarbon having the number of carbon atoms designated (i.e. C₁₋₆means one to six carbon atoms) and including straight, branched chain,or cyclic substituent groups. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, andcyclopropylmethyl.

As used herein, the term “substituted alkyl” means alkyl as definedabove, substituted by one, two or three substituents selected from thegroup consisting of halogen, —OH, alkoxy, —NH₂, amino, azido, —N(CH₃)₂,—C(═O)OH, trifluoromethyl, —C(═O)O(C₁-C₄)alkyl, —C(═O)NH₂, —SO₂NH₂,—C(═NH)NH₂, and —NO₂. Examples of substituted alkyls include, but arenot limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl and3-chloropropyl.

As used herein, the term “heteroalkyl” by itself or in combination withanother term means, unless otherwise stated, a stable straight orbranched chain alkyl group consisting of the stated number of carbonatoms and one or two heteroatoms selected from the group consisting of0, N, and S, and wherein the nitrogen and sulfur atoms may be optionallyoxidized and the nitrogen heteroatom may be optionally quaternized. Theheteroatom(s) may be placed at any position of the heteroalkyl group,including between the rest of the heteroalkyl group and the fragment towhich it is attached, as well as attached to the most distal carbon atomin the heteroalkyl group. Examples include:

-   -   —O—CH₂—CH₂—CH₃, —CH₂—CH₂—CH₂—OH, —CH₂—CH₂—NH—CH₃,        —CH₂—S—CH₂—CH₃, and —CH₂CH₂—S(═O)—CH₃. Up to two heteroatoms may        be consecutive, such as, for example, —CH₂—NH—OCH₃, or        —CH₂—CH₂—S—S—CH₃

As used herein, the term “alkoxy” employed alone or in combination withother terms means, unless otherwise stated, an alkyl group having thedesignated number of carbon atoms, as defined above, connected to therest of the molecule via an oxygen atom, such as, for example, methoxy,ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs andisomers.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom.

As used herein, the term “cycloalkyl” refers to a mono cyclic orpolycyclic non-aromatic radical, wherein each of the atoms forming thering (i.e. skeletal atoms) is a carbon atom. In one embodiment, thecycloalkyl group is saturated or partially unsaturated. In anotherembodiment, the cycloalkyl group is fused with an aromatic ring.Cycloalkyl groups include groups having from 3 to 10 ring atoms.Illustrative examples of cycloalkyl groups include, but are not limitedto, the following moieties:

Monocyclic cycloalkyls include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.Dicyclic cycloalkyls include, but are not limited to,tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycycliccycloalkyls include adamantine and norbornane. The term cycloalkylincludes “unsaturated nonaromatic carbocyclyl” or “nonaromaticunsaturated carbocyclyl” groups, both of which refer to a nonaromaticcarbocycle as defined herein, which contains at least one carbon doublebond or one carbon triple bond.

As used herein, the term “heterocycloalkyl” or “heterocyclyl” refers toa cyclic group containing one to four ring heteroatoms each selectedfrom O, S, and N. In one embodiment, each heterocycloalkyl group hasfrom 4 to 10 atoms in its ring system, with the proviso that the ring ofsaid group does not contain two adjacent 0 atoms. In another embodiment,the heterocycloalkyl group is fused with an aromatic ring. In oneembodiment, the nitrogen and sulfur heteroatoms may be optionallyoxidized, and the nitrogen atom may be optionally quaternized. Theheterocyclic system may be attached, unless otherwise stated, at anyheteroatom or carbon atom that affords a stable structure. A heterocyclemay be aromatic or non-aromatic in nature. In one embodiment, theheterocycle is a heteroaryl.

An example of a 3-membered heterocycloalkyl group includes, and is notlimited to, aziridine. Examples of 4-membered heterocycloalkyl groupsinclude, and are not limited to, azetidine and a beta lactam. Examplesof 5-membered heterocycloalkyl groups include, and are not limited to,pyrrolidine, oxazolidine and thiazolidinedione. Examples of 6-memberedheterocycloalkyl groups include, and are not limited to, piperidine,morpholine and piperazine.

Other non-limiting examples of heterocycloalkyl groups are:

Examples of non-aromatic heterocycles include monocyclic groups such asaziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane,2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran,1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin, and hexamethyleneoxide.

As used herein, the term “aromatic” refers to a carbocycle orheterocycle with one or more polyunsaturated rings and having aromaticcharacter, i.e. having (4n+2) delocalized π (pi) electrons, where n isan integer.

As used herein, the term “aryl,” employed alone or in combination withother terms, means, unless otherwise stated, a carbocyclic aromaticsystem containing one or more rings (typically one, two or three rings),wherein such rings may be attached together in a pendent manner, such asa biphenyl, or may be fused, such as naphthalene. Examples of arylgroups include phenyl, anthracyl, and naphthyl.

As used herein, the term “aryl-(C₁-C₃)alkyl” means a functional groupwherein a one- to three-carbon alkylene chain is attached to an arylgroup, e.g., —CH₂CH₂— phenyl, —CH₂-phenyl (benzyl), aryl-CH₂— andaryl-CH(CH₃)—. The term “substituted aryl-(C₁-C₃)alkyl” means anaryl-(C₁-C₃)alkyl functional group in which the aryl group issubstituted. Similarly, the term “heteroaryl-(C₁-C₃)alkyl” means afunctional group wherein a one to three carbon alkylene chain isattached to a heteroaryl group, e.g., —CH₂CH₂-pyridyl. The term“substituted heteroaryl-(C₁-C₃)alkyl” means a heteroaryl-(C₁-C₃)alkylfunctional group in which the heteroaryl group is substituted.

As used herein, the term “heteroaryl” or “heteroaromatic” refers to aheterocycle having aromatic character. A polycyclic heteroaryl mayinclude one or more rings that are partially saturated. Examples includethe following moieties:

Examples of heteroaryl groups also include pyridyl, pyrazinyl,pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl,furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl,oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and1,3,4-oxadiazolyl.

Examples of polycyclic heterocycles and heteroaryls include indolyl(particularly 3-, 4-, 6- and 7-indolyl), indolinyl, quinolyl,tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl),1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2-and quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl,coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (particularly3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl,1,2-benzisoxazolyl, benzothienyl (particularly 3-, 4-, 5-, 6-, and7-benzothienyl), benzoxazolyl, benzothiazolyl (particularly2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl(particularly 2-benzimidazolyl), benzotriazolyl, thioxanthinyl,carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.

As used herein, the term “substituted” means that an atom or group ofatoms has replaced hydrogen as the substituent attached to anothergroup. The term “substituted” further refers to any level ofsubstitution, namely mono-, di-, tri-, tetra-, or penta-substitution,where such substitution is permitted. The substituents are independentlyselected, and substitution may be at any chemically accessible position.In one embodiment, the substituents vary in number between one and four.In another embodiment, the substituents vary in number between one andthree. In yet another embodiment, the substituents vary in numberbetween one and two.

As used herein, the term “optionally substituted” means that thereferenced group may be substituted or unsubstituted. In one embodiment,the referenced group is optionally substituted with zero substituents,i.e., the referenced group is unsubstituted. In another embodiment, thereferenced group is optionally substituted with one or more additionalgroup(s) individually and independently selected from groups describedherein.

In one embodiment, the substituents are independently selected from thegroup consisting of oxo, halogen, —CN, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂,alkyl (including straight chain, branched and/or unsaturated alkyl),substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, fluoro alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy,—S-alkyl, S(═O) 2 alkyl, S(═O) 2 N[H, alkyl, or aryl],—C(═O)NH[substituted or unsubstituted alkyl, or substituted orunsubstituted phenyl], —C(═O)N[H or substituted or unsubstituted alkylor aryl]₂, —OC(═O)N[substituted or unsubstituted alkyl]₂,—NHC(═O)NH[substituted or unsubstituted alkyl, or substituted orunsubstituted phenyl], —NHC(═O)alkyl, —N[substituted or unsubstitutedalkyl]C(═O)[substituted or unsubstituted alkyl], —NHC(═O)[substituted orunsubstituted alkyl], —C(OH)[substituted or unsubstituted alkyl]₂, and—C(NH₂)[substituted or unsubstituted alkyl]₂. In another embodiment, byway of example, an optional substituent is selected from oxo, fluorine,chlorine, bromine, iodine, —CN, —NH 2, —OH, —NH(CH₃), —N(CH₃)₂, —CH₃,—CH₂CH₃, —CH(CH₃)₂, —CF₃, —CH₂CF₃, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —OCF₃,—OCH₂CF₃, —S(═O)₂—CH₃, —C(═O)NH₂, —C(═O)—NHCH₃, —NHC(═O)NHCH₃,—C(═O)CH₃, —ON(O)₂, and —C(═O)OH. In yet one embodiment, thesubstituents are independently selected from the group consisting ofC₁₋₆ alkyl, —OH, C₁₋₆ alkoxy, halo, amino, acetamido, oxo and nitro. Asused herein, where a substituent is an alkyl or alkoxy group, the carbonchain may be branched, straight or cyclic.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

In eukaryotes, the self-splicing group II intron is a RNA tertiarystructure that is absent in vertebrates but essential for respiration inplants, fungi, yeast, bacteria, ichthyosporea and worms. In yeast andsome fungi, it is located in mitochondria and it interrupts housekeepinggenes essential for respiration. In plants, group II introns can belocated in genes within the chloroplast and/or the mitochondrial genome,where they frequently interrupt genes important for respiration andother metabolic functions. Without being bound by any particular theory,it is believed that group II introns are important for gene expressionin a diversity of unicellular and multicellular eukaryotic organisms,including parasites. The invention is based, in part, on the discoverythat inhibition of group II introns results in the growth inhibition ofpathogenic and nonpathogenic yeast species and that high affinitycompounds can be generated that specifically, inhibit group II intronsplicing in-vitro and in-vivo and that lack toxicity in human cells. Forexample, it is demonstrated herein that the compounds are potent growthinhibitors of the pathogen Candida parapsilosis, displaying antifungalactivity comparable with amphotericin B.

In one embodiment, the present invention relates to compositions andmethods for inhibiting group II introns, including inhibiting group IIintron splicing. In some embodiments, the invention relates topreventing, inhibiting, or disrupting the growth of an organismharboring an active group II intron (i.e., a group II intron that iscapable of self-splicing).

In some embodiments, the invention relates to treating or preventing adisease or disorder associated with an organism harboring an activegroup II intron. In one embodiment, the group II intron splicinginhibitor of the invention is used as a therapeutic agent for treatmentof a disease or disorder. In one embodiment, the disease or disorder isselected from the group consisting of a fungal infection, a yeastinfection, and a bacterial infection. In one embodiment, the disease ordisorder is associated with a fungal infection, a yeast infection, or abacterial infection.

In one embodiment, the present invention relates to compositions andmethods for reducing or preventing the growth of an organism thatharbors a group II intron. Therefore, in one embodiment, the group IIintron splicing inhibitor of the invention is used to reduce or preventthe growth of a plant, a fungus, a yeast, a bacteria or a protist.

In one embodiment, the present invention provides a compositioncomprising an inhibitor of group II intron splicing. In one embodiment,the inhibitor of group II intron splicing is selected from a protein, apeptide, a peptidomimetic, an antibody, a ribozyme, a small moleculechemical compound, a nucleic acid, a vector, and an antisense nucleicacid molecule.

In one embodiment, the inhibitor of group II intron splicing is a smallmolecule chemical compound. Exemplary small molecule chemical compoundsinclude aurones, 2-benzoylbenzofurans, and benzoyl hydrazones.

In one embodiment, the invention provides methods for treating orpreventing a disease or disorder associated with an organism harboringan active group II intron. In one embodiment, the method comprisesadministering a composition comprising an inhibitor of group II intronsplicing to a subject in need thereof. In one embodiment, the subject isa vertebrate animal. In another embodiment, the vertebrate animal is ahuman.

In one embodiment, the invention provides methods for reducing growth ofan organism harboring an active group II intron. In one embodiment, themethod comprises contacting an organism harboring an active group IIintron with a composition comprising an inhibitor of group II intronsplicing.

In one embodiment, the invention provides methods for preventing growthof an organism harboring an active group II intron on a surface orsubstrate. In one embodiment, the method comprises contacting a surfaceor substrate with a composition comprising an inhibitor of group IIintron splicing.

In one embodiment, the inhibitor of group II intron splicing is at leastone of the group consisting of a chemical compound, a protein, apeptide, a peptidomimetic, an antibody, a ribozyme, a small moleculechemical compound, a nucleic acid, a vector, an antisense nucleic acidmolecule.

Inhibitors

In one embodiment, the present invention provides a compositioncomprising an inhibitor of group II intron splicing. In one embodiment,an inhibitor of group II intron splicing is any compound, molecule, oragent that reduces, inhibits, or prevents the self-splicing or ribozymefunction of group II introns. In one embodiment, an inhibitor of groupII intron splicing comprises a nucleic acid, a peptide, a small moleculechemical compound, a siRNA, a ribozyme, an antisense nucleic acid, anantagonist, an aptamer, a peptidomimetic, or any combination thereof.

Small Molecule Inhibitors

In various embodiments, the inhibitor is a small molecule chemicalcompound, including, but limited to, an aurone, a 2-benzoylbenzofuran,or a benzoyl hydrazone.

In one embodiment, the small molecule chemical compound is a compound ofFormula (I) or a salt thereof;

wherein in Formula (I):

-   -   X is O, S, or NR¹⁰;    -   R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each        independently selected from the group consisting of H, —C₁-C₆        alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, aryl, heteroaryl,        cycloalkyl, heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂, —OH,        —OR¹¹, —SR¹¹, —S(═O)R¹¹, —S(═O)₂R¹¹, —NHS(═O)₂R¹¹, —C(═O)R¹¹,        —OC(═O)R¹¹, —CO₂R¹¹, —OCO₂R¹¹, —CH(R¹¹)₂, —N(R¹¹)₂,        —C(═O)N(R¹¹)₂, —C(═O)NHR¹¹, —OC(═O)N(R¹¹)₂, —NHC(═O)NH(R¹¹),        —NHC(═O)R¹¹, —NHC(═O)OR¹¹, —C(OH)(R¹¹)₂, and —C(NH₂)(R¹¹)₂, and        combinations thereof    -   or optionally two adjacent R¹-R⁵ or R⁶-R⁹ are joined to form a        ring;    -   each occurrence of R¹¹ is independently selected from the group        consisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆        heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, F,        Cl, Br, I, —CN, —NO₂, —OH, and combinations thereof; and    -   wherein only one of the two following conditions is met:        -   condition 1, wherein the bond between carbons 1 and 2 is a            single bond, the bond between carbons 2 and 3 is a double            bond, Y is (═O), and Z is H; or        -   condition 2, wherein the bond between carbons 1 and 2 is a            double bond, the bond between carbons 2 and 3 is a single            bond, Y is H, and Z is (═O).

In one embodiment, the small molecule chemical compound of Formula (I)is a compound of Formula (II):

In one embodiment, the compound of Formula (I) is a compound of Formula(III):

In one embodiment, R², R³, and R⁴ are OH. In one embodiment, R⁶ and R⁸are OH. In one embodiment, R⁷ and R⁹ are Cl.

In one embodiment, R⁷ is represented by Formula (IV):

-   -   wherein * represents the attachment to Formula (I), (II), or        (III);    -   A¹, A², A³, and A⁴ are CR¹³ or N;    -   R¹² is selected from the group consisting of H, C₁-C₆ alkyl,        C₁-C₆ fluoroalkyl, C₁-C₆ perfluoroalkyl, C₁-C₆ heteroalkyl,        aryl, heteroaryl, cycloalkyl, heterocycloalkyl, F, Cl, Br, I,        —CN, —NO₂, —OH, —OR¹⁴, —SR¹⁴, —S(═O)R¹⁴, —S(═O)₂R¹⁴,        —S(═O)₂NHR¹⁴, —S(═O)₂N(R¹⁴)₂, —NHS(═O)₂R¹⁴, —C(═O)R¹⁴,        —OC(═O)R¹⁴, —CO₂R¹⁴, —OCO₂R¹⁴, —CH(R¹⁴)₂, —N(R¹⁴)₂,        —C(═O)N(R¹⁴)₂, —C(═O)NHR¹⁴, —OC(═O)N(R¹⁴)₂, —NHC(═O)NH(R¹⁴),        —NHC(═O)R¹⁴, —NHC(═O)OR¹⁴, —C(OH)(R¹⁴)₂, and —C(NH₂)(R¹⁴)₂, and        combinations thereof;    -   each occurrence of R¹³ is independently selected from the group        consisting of H, C₁-C₆ alkyl, F, Cl, Br, I, —CN, —NO₂, —OH,        —CF₃, C₁-C₆ heteroalkyl, aryl-(C₁-C₃)alkyl, and cycloalkyl; or        optionally two adjacent R¹³ are joined to form a ring;    -   each occurrence of R¹⁴ is independently selected from the group        consisting of H, C₁-C₆ alkyl, F, Cl, Br, I, —CN, —NO₂, —OH,        —CF₃, C₁-C₆ heteroalkyl, aryl-(C₁-C₃)alkyl, and cycloalkyl; or        optionally two R¹⁴ on the same atom may together form a ring.

In one embodiment, A¹, A², A³, and A⁴ are CR¹³. In one embodiment, A¹,A³, and A⁴ are CR¹³ and A² is N. In one embodiment, A¹, A², and A⁴ areCR¹³ and A³ is N. In one embodiment, A¹ and A⁴ are CR¹³ and A² and A³are N.

In one embodiment, R⁸ is —C(═O)NHR¹⁰.

In one embodiment, the compound of Formula (I) is selected from thegroup consisting of:

In another aspect, the small molecule chemical compound is a compound ofFormula (V) or a salt thereof;

wherein in Formula (V):

-   -   L is a divalent linking group selected from the group consisting        of a single bond and ethylene;    -   R²¹, R²², R²³, R²⁴, and R²⁵ are each independently selected from        the group consisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl,        —C₁-C₆ heteroalkyl, aryl, heteroaryl, cycloalkyl,        heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂, —OH, —SR²⁷,        —S(═O)R²⁷, —S(═O)₂R²⁷, —NHS(═O)₂R²⁷, —C(═O)R²⁷, —OC(═O)R²⁷,        —CO₂R²⁷, —OCO₂R²⁷, —CH(R²⁷)₂, —N(R²⁷)₂, —C(═O)N(R²⁷)₂,        —C(═O)NHR²⁷, —OC(═O)N(R²⁷)₂, —NHC(═O)NH(R²⁷), —NHC(═O)R²⁷,        —NHC(═O)OR²⁷, —C(OH)(R²⁷)₂, and —C(NH₂)(R²⁷)₂, and combinations        thereof;    -   or optionally two adjacent R²¹-R²⁵ are joined to form a ring;    -   each occurrence of R²⁷ is independently selected from the group        consisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆        heteroalkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, F,        Cl, Br, I, —CN, —NO₂, —OH, and combinations thereof; and    -   R²⁶ is selected from the group consisting of an aryl group and a        heteroaryl group, wherein the aryl or heteroaryl group may be        optionally substituted.

In one embodiment, R²², R²³, and R²⁴ are OH.

In one embodiment, R²⁶ is an aryl group which may be optionallysubstituted. In one embodiment, R²⁶ is a phenyl group. In oneembodiment, R²⁶ is a naphthyl group. In another embodiment, R²⁶ is aheteroaryl group which may be optionally substituted. In one embodiment,R²⁶ is a furyl group. In one embodiment, R²⁶ is a pyridinyl group. Inone embodiment, R²⁶ is a diazenyl group.

In one embodiment, R²⁶ is a group of Formula (VI):

wherein in Formula (VI):

-   -   * represents the attachment to Formula (V);    -   Z¹, Z², Z³, Z⁴, and Z⁵ are CR²⁸ or N;    -   each occurrence of R²⁸ is independently selected from the group        consisting of H, C₁-C₆ alkyl, F, Cl, Br, I, —CN, —NO₂, —OH,        —CF₃, —OR²⁹, —N(R²⁹)₂, —C(═O)R²⁹, C₁-C₆ heteroalkyl,        aryl-(C₁-C₃)alkyl, cycloalkyl, alkynyl, and combinations        thereof; or optionally two adjacent R²⁸ are joined together to        form a ring; and    -   R²⁹ is selected from the group consisting of H, C₁-C₆ alkyl, or        heteroaryl-(C₁-C₃)alkyl.

In one embodiment, the compound of Formula (V) is selected from thegroup consisting of:

When the inhibitor is a small molecule, a small molecule may be obtainedusing standard methods known to the skilled artisan. Such methodsinclude chemical organic synthesis or biological means. Biological meansinclude purification from a biological source, recombinant synthesis andin vitro translation systems, using methods well known in the art. Inone embodiment, a small molecule inhibitor of the invention comprises anorganic molecule, inorganic molecule, biomolecule, synthetic molecule,and the like.

Combinatorial libraries of molecularly diverse chemical compounds arewell known in the art as are method of making the libraries. The methodmay use a variety of techniques well-known to the skilled artisanincluding solid phase synthesis, solution methods, parallel synthesis ofsingle compounds, synthesis of chemical mixtures, rigid core structures,flexible linear sequences, deconvolution strategies, tagging techniques,and generating unbiased molecular landscapes for lead discovery vs.biased structures for lead development.

In a general method for small library synthesis, an activated coremolecule is condensed with a number of building blocks, resulting in acombinatorial library of covalently linked, core-building blockensembles. The shape and rigidity of the core determines the orientationof the building blocks in shape space. The libraries can be biased bychanging the core, linkage, or building blocks to target a characterizedbiological structure (“focused libraries”) or synthesized with lessstructural bias using flexible cores.

The small molecule and small molecule compounds described herein may bepresent as salts even if salts are not depicted and it is understoodthat the invention embraces all salts and solvates of the inhibitorsdepicted here, as well as the non-salt and non-solvate form of theinhibitors, as is well understood by the skilled artisan. In someembodiments, the salts of the inhibitors of the invention arepharmaceutically acceptable salts.

Where tautomeric forms may be present for any of the inhibitorsdescribed herein, each and every tautomeric form is intended to beincluded in the present invention, even though only one or some of thetautomeric forms may be explicitly depicted. For example, when a2-hydroxypyridyl moiety is depicted, the corresponding 2-pyridonetautomer is also intended.

The invention also includes any or all of the stereochemical forms,including any enantiomeric or diasteriomeric forms of the inhibitorsdescribed. The recitation of the structure or name herein is intended toembrace all possible stereoisomers of inhibitors depicted. All forms ofthe inhibitors are also embraced by the invention, such as crystallineor non-crystalline forms of the inhibitors. Compositions comprising aninhibitor of the invention are also intended, such as a composition ofsubstantially pure inhibitor, including a specific stereochemical formthereof, or a composition comprising mixtures of inhibitors of theinvention in any ratio, including two or more stereochemical forms, suchas in a racemic or non-racemic mixture.

In one embodiment, the small molecule inhibitor of the inventioncomprises an analog or derivative of an inhibitor described herein.

In one embodiment, the small molecules described herein are candidatesfor derivatization. As such, in some instances, the analogs of the smallmolecules described herein that have modulated potency, selectivity, andsolubility are included herein and provide useful leads for drugdiscovery and drug development. Thus, in some instances, duringoptimization new analogs are designed considering issues of drugdelivery, metabolism, novelty, and safety.

In some instances, small molecule inhibitors described herein arederivatized/analogued as is well known in the art of combinatorial andmedicinal chemistry. The analogs or derivatives can be prepared byadding and/or substituting functional groups at various locations. Assuch, the small molecules described herein can be converted intoderivatives/analogs using well known chemical synthesis procedures. Forexample, all of the hydrogen atoms or substituents can be selectivelymodified to generate new analogs. Also, the linking atoms or groups canbe modified into longer or shorter linkers with carbon backbones orhetero atoms. Also, the ring groups can be changed so as to have adifferent number of atoms in the ring and/or to include hetero atoms.Moreover, aromatics can be converted to cyclic rings, and vice versa.For example, the rings may be from 5-7 atoms, and may be homocycles orheterocycles.

As used herein, the term “analog,” “analogue,” or “derivative” is meantto refer to a chemical compound or molecule made from a parent compoundor molecule by one or more chemical reactions. As such, an analog can bea structure having a structure similar to that of the small moleculeinhibitors described herein or can be based on a scaffold of a smallmolecule inhibitor described herein, but differing from it in respect tocertain components or structural makeup, which may have a similar oropposite action metabolically. An analog or derivative of any of a smallmolecule inhibitor in accordance with the present invention can be usedto inhibit group II introns.

In one embodiment, the small molecule inhibitors described herein canindependently be derivatized/analoged by modifying hydrogen groupsindependently from each other into other substituents. That is, eachatom on each molecule can be independently modified with respect to theother atoms on the same molecule. Any traditional modification forproducing a derivative/analog can be used. For example, the atoms andsubstituents can be independently comprised of hydrogen, an alkyl,aliphatic, straight chain aliphatic, aliphatic having a chain heteroatom, branched aliphatic, substituted aliphatic, cyclic aliphatic,heterocyclic aliphatic having one or more hetero atoms, aromatic,heteroaromatic, polyaromatic, polyamino acids, peptides, polypeptides,combinations thereof, halogens, halo-substituted aliphatics, and thelike. Additionally, any ring group on a compound can be derivatized toincrease and/or decrease ring size as well as change the backbone atomsto carbon atoms or hetero atoms.

Nucleic Acid Inhibitors

In some embodiments, the invention includes nucleic acid molecules andgene therapy agents that can inhibit group II intron splicing.

In some instances the inhibitor is an antisense molecule or aptamer,which inhibits a group II intron RNA molecule. Domain V of a group IIintron is the most highly conserved domain, and it is indispensable forsplicing. Therefore, in one embodiment, an antisense molecule of theinvention targets domain V of a group II intron. In one embodiment, thenucleic acid comprises a promoter/regulatory sequence such that thepromoter/regulatory sequence is capable of directing expression of thenucleic acid or increasing or decreasing stability of the nucleic acid.Thus, the invention encompasses expression vectors and methods for theintroduction of exogenous DNA into cells with concomitant expression ofthe exogenous DNA in the cells such as those described, for example, inSambrook et al. (2012, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York), and in Ausubel et al. (1997,Current Protocols in Molecular Biology, John Wiley & Sons, New York) andas described elsewhere herein.

In one embodiment, siRNA is used to inhibit a group II intron RNAmolecule. RNA interference (RNAi) is a phenomenon in which theintroduction of double-stranded RNA (dsRNA) into a diverse range oforganisms and cell types causes degradation of the complementary mRNA.In the cell, long dsRNAs are cleaved into short 21-25 nucleotide smallinterfering RNAs, or siRNAs, by a ribonuclease known as Dicer. ThesiRNAs subsequently assemble with protein components into an RNA-inducedsilencing complex (RISC), unwinding in the process. Activated RISC thenbinds to complementary transcript by base pairing interactions betweenthe siRNA antisense strand and the mRNA. The bound mRNA is cleaved andsequence specific degradation of mRNA results in gene silencing. See,for example, U.S. Pat. No. 6,506,559; Fire et al., 1998, Nature391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery etal., 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference(RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, P A(2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutscheket al. (2004, Nature 432:173-178) describe a chemical modification tosiRNAs that aids in intravenous systemic delivery. Optimizing siRNAsinvolves consideration of overall G/C content, C/T content at thetermini, Tm and the nucleotide content of the 3′ overhang. See, forinstance, Schwartz et al., 2003, Cell, 115:199-208 and Khvorova et al.,2003, Cell 115:209-216. Therefore, the present invention also includesmethods of inhibiting group II intron splicing using RNAi technology.

In another embodiment, the invention includes a vector comprising ansiRNA or antisense polynucleotide. In one embodiment, the siRNA orantisense polynucleotide is capable of inhibiting group II intronsplicing. The incorporation of a desired polynucleotide into a vectorand the choice of vectors is well-known in the art as described in, forexample, Sambrook et al. (2012), and in Ausubel et al. (1997), andelsewhere herein.

In some embodiments, the expression vectors described herein encode ashort hairpin RNA (shRNA) inhibitor. shRNA inhibitors are well known inthe art and are directed against the mRNA of a target, therebydecreasing the expression of the target. In some embodiments, theencoded shRNA is expressed by a cell, and is then processed into siRNA.For example, in some instances, the cell possesses native enzymes (e.g.,dicer) that cleaves the shRNA to form siRNA.

The siRNA, shRNA, or antisense polynucleotide can be cloned into anumber of types of vectors as described elsewhere herein. For expressionof the siRNA or antisense polynucleotide, at least one module in eachpromoter functions to position the start site for RNA synthesis.

In order to assess the expression of the siRNA, shRNA, or antisensepolynucleotide, the expression vector to be introduced into a cell canalso contain either a selectable marker gene or a reporter gene or bothto facilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected using a viralvector. In other embodiments, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers are known in the art and include, for example,antibiotic-resistance genes, such as neomycin resistance and the like.

Therefore, in another embodiment, the invention relates to a vector,comprising the nucleotide sequence of the invention or the construct ofthe invention. The choice of the vector will depend on the host cell inwhich it is to be subsequently introduced. In a particular embodiment,the vector of the invention is an expression vector. Suitable host cellsinclude a wide variety of prokaryotic and eukaryotic host cells. Inspecific embodiments, the expression vector is selected from the groupconsisting of a viral vector, a bacterial vector and a mammalian cellvector. Prokaryote- and/or eukaryote-vector based systems can beemployed for use with the present invention to produce polynucleotides,or their cognate polypeptides. Many such systems are commercially andwidely available.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2012), and in Ausubel et al.(1997), and in other virology and molecular biology manuals. Viruses,which are useful as vectors include, but are not limited to,retroviruses, adenoviruses, adeno-associated viruses, herpes viruses,and lentiviruses. In general, a suitable vector contains an origin ofreplication functional in at least one organism, a promoter sequence,convenient restriction endonuclease sites, and one or more selectablemarkers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193.

By way of illustration, the vector in which the nucleic acid sequence isintroduced can be a plasmid which is or is not integrated in the genomeof a host cell when it is introduced in the cell. Illustrative,non-limiting examples of vectors in which the nucleotide sequence of theinvention or the gene construct of the invention can be inserted includea tet-on inducible vector for expression in eukaryote cells.

The vector may be obtained by conventional methods known by personsskilled in the art (Sambrook et al., 2012). In a particular embodiment,the vector is a vector useful for transforming animal cells.

In one embodiment, the recombinant expression vectors may also containnucleic acid molecules which encode a peptide or peptidomimeticinhibitor of invention, described elsewhere herein.

A promoter may be one naturally associated with a gene or polynucleotidesequence, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment and/or exon. Such a promoter canbe referred to as “endogenous.” Similarly, an enhancer may be onenaturally associated with a polynucleotide sequence, located eitherdownstream or upstream of that sequence. Alternatively, certainadvantages will be gained by positioning the coding polynucleotidesegment under the control of a recombinant or heterologous promoter,which refers to a promoter that is not normally associated with apolynucleotide sequence in its natural environment. A recombinant orheterologous enhancer refers also to an enhancer not normally associatedwith a polynucleotide sequence in its natural environment. Suchpromoters or enhancers may include promoters or enhancers of othergenes, and promoters or enhancers isolated from any other prokaryotic,viral, or eukaryotic cell, and promoters or enhancers not “naturallyoccurring,” i.e., containing different elements of differenttranscriptional regulatory regions, and/or mutations that alterexpression. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (U.S. Pat. Nos.4,683,202 and 5,928,906). Furthermore, it is contemplated the controlsequences that direct transcription and/or expression of sequenceswithin non-nuclear organelles such as mitochondria, chloroplasts, andthe like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the celltype, organelle, and organism chosen for expression. Those of skill inthe art of molecular biology generally know how to use promoters,enhancers, and cell type combinations for protein expression, forexample, see Sambrook et al. (2012). The promoters employed may beconstitutive, tissue-specific, inducible, and/or useful under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The promoter may be heterologousor endogenous.

The recombinant expression vectors may also contain a selectable markergene which facilitates the selection of transformed or transfected hostcells. Suitable selectable marker genes are genes encoding proteins suchas G418 and hygromycin which confer resistance to certain drugs,β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase,or an immunoglobulin or portion thereof such as the Fc portion of animmunoglobulin (e.g., IgG). The selectable markers may be introduced ona separate vector from the nucleic acid of interest.

Following the generation of the siRNA polynucleotide, a skilled artisanwill understand that the siRNA polynucleotide will have certaincharacteristics that can be modified to improve the siRNA as atherapeutic compound. Therefore, the siRNA polynucleotide may be furtherdesigned to resist degradation by modifying it to includephosphorothioate, or other linkages, methylphosphonate, sulfone,sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters,and the like (see, e.g., Agrwal et al., 1987, Tetrahedron Lett.28:3539-3542; Stec et al., 1985 Tetrahedron Lett. 26:2191-2194; Moody etal., 1989 Nucleic Acids Res. 12:4769-4782; Eckstein, 1989 Trends Biol.Sci. 14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitorsof Gene Expression, Cohen, ed., Macmillan Press, London, pp. 97-117(1989)).

Any polynucleotide may be further modified to increase its stability invivo. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends; the use ofphosphorothioate or 2′ O-methyl rather than phosphodiester linkages inthe backbone; and/or the inclusion of nontraditional bases such asinosine, queuosine, and wybutosine and the like, as well as acetyl-methyl-, thio- and other modified forms of adenine, cytidine, guanine,thymine, and uridine.

Antisense molecules and their use for inhibiting RNA molecules are wellknown in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides,Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleicacids are DNA or RNA molecules that are complementary, as that term isdefined elsewhere herein, to at least a portion of a specific mRNAmolecule (Weintraub, 1990, Scientific American 262:40). In the cell,antisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule thereby inhibiting the translation of genes.

In one embodiment, antisense molecules of the invention may be madesynthetically. In one embodiment, antisense oligomers of between about10 to about 30 nucleotides are used, since they are easily synthesizedfor administration to a subject or a target cell. Synthetic antisensemolecules contemplated by the invention include oligonucleotidederivatives known in the art which have improved biological activitycompared to unmodified oligonucleotides (see U.S. Pat. No. 5,023,243).

In one embodiment of the invention, a ribozyme is used to inhibit groupII intron splicing. Ribozymes useful for inhibiting a target moleculemay be designed by incorporating target sequences into the basicribozyme structure which are complementary, for example, to the mRNAsequence of a group II intron. Ribozymes targeting group II introns maybe synthesized using commercially available reagents (Glen ResearchCorp., Sterling, VA, or BioAutomation, Plano, TX) or they may begenetically expressed from DNA encoding them.

Genome Editing System

In one embodiment, the invention provides for inhibition of group IIintron splicing through use of a genome editing system. A series ofprogrammable nuclease-based genome editing technologies have developed(see for example, Hsu et al., Cell 157, Jun. 5, 2014 1262-1278),including, but not limited to, meganucleases, zinc finger nucleases(ZFNs), transcription activator-like effector-based nucleases (TALENs)and CRISPR-Cas systems (see e.g. Platt et al., Cell 159(2), 440-455(2014); Shalem et al., Science 3 84-87 (2014); and Le Cong et al.,Science 339, 819 (2013)) or alternative CRISPR systems. Genome editingsystems have a wide variety of utilities including modifying (e.g.,deleting, inserting, translocating, inactivating, activating,repressing, altering methylation, transferring specific moieties) atarget polynucleotide in a multiplicity of cell types.

In one embodiment, a CRISPR-Cas system, where a guide RNA (gRNA)targeted to a nucleic acid molecule harboring an active group II intron,and a CRISPR-associated (Cas) peptide form a complex to induce mutationswithin the targeted intron sequence. The CRISPR complex of the inventionprovides an effective means for modifying a target polynucleotide.

In one embodiment, the composition of the present invention comprises aCas peptide or Cas-derived peptide and a gRNA targeted to a group IIintron or to a DNA sequence encoding a group II intron. In oneembodiment, the composition comprises a nucleic acid molecule encoding aCas peptide or Cas-derived peptide. In one embodiment, the compositioncomprises a nucleic acid molecule encoding a gRNA targeted to a group IIintron or to a DNA sequence encoding a group II intron.

In one embodiment, the target polynucleotide is a DNA molecule. DNAmolecules include, but are not limited to, genomic DNA molecules,extrachromosomal DNA molecules, conjugative plasmids and exogenous DNAmolecules. In one embodiment, the target polynucleotide is a RNAmolecule (see e.g., Batra et al., Cell. 170(5):899-912.e10 (2017) formethods of use of a CRISPR-Cas system to modify an RNA molecule.)

In general, “CRISPR-Cas system” or “CRISPR system” refers collectivelyto transcripts and other elements involved in the expression of ordirecting the activity of CRISPR-associated (“Cas”) genes, includingsequences encoding a Cas gene, a tracr (trans-activating CRISPR)sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-matesequence (encompassing a “direct repeat” and a tracrRNA-processedpartial direct repeat in the context of an endogenous CRISPR system), aguide sequence (also referred to as a “spacer” in the context of anendogenous CRISPR system), or other sequences and transcripts from aCRISPR locus. In some embodiments, one or more elements of a CRISPRsystem is derived from a type I, type II, or type III CRISPR system. Insome embodiments, one or more elements of a CRISPR system are derivedfrom a particular organism comprising an endogenous CRISPR system, suchas Streptococcus pyogenes. In general, a CRISPR system is characterizedby elements that promote the formation of a CRISPR complex at the siteof a target sequence (also referred to as a protospacer in the contextof an endogenous CRISPR system).

In some embodiments, the site of nuclease activity is determined by theCRISPR-Cas system guide RNA. In general, a “CRISPR-Cas guide RNA” or“guide RNA” refers to an RNA that directs sequence-specific binding of aCRISPR complex to the target sequence. Typically, a guide RNA comprises(i) a guide sequence that has sufficient complementarity with a targetpolynucleotide sequence to hybridize with the target sequence and (ii) atrans-activating cr (tracr) mate sequence. In some embodiments, thedegree of complementarity between a guide sequence and its correspondingtarget sequence, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. In some embodiments, a guide sequence is aboutor more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or morenucleotides in length. In some embodiments, a guide sequence is lessthan about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotidesin length. The ability of a guide sequence to direct sequence-specificbinding of a CRISPR complex to a target sequence may be assessed by anysuitable assay. For example, the components of a CRISPR systemsufficient to form a CRISPR complex, including the guide sequence to betested, may be provided to a host cell having the corresponding targetsequence, such as by transfection with vectors encoding the componentsof the CRISPR sequence, followed by an assessment of preferentialcleavage within the target sequence, such as by Surveyor assay asdescribed herein. Similarly, cleavage of a target polynucleotidesequence may be evaluated in a test tube by providing the targetsequence, components of a CRISPR complex, including the guide sequenceto be tested and a control guide sequence different from the test guidesequence, and comparing binding or rate of cleavage at the targetsequence between the test and control guide sequence reactions. Otherassays are possible, and will occur to those skilled in the art.

In the context of formation of a CRISPR complex, a “target sequence” or“a sequence of a target DNA” refers to a sequence to which a guidesequence is designed to have complementarity, where hybridizationbetween a target sequence and a guide sequence promotes the formation ofa CRISPR complex. Full complementarity is not necessarily required,provided there is sufficient complementarity to cause hybridization andpromote formation of a CRISPR complex. A target sequence may compriseany polynucleotide, such as DNA or RNA polynucleotides or DNA/RNA hybridpolynucleotides. In some embodiments, a target sequence is located inthe nucleus or cytoplasm of a cell. In some embodiments, the targetsequence may be within an organelle of a eukaryotic cell, for example,mitochondrion or chloroplast.

In some embodiments, the CRISPR-Cas domain comprises a Cas protein.Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3,Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2. Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4,homologs thereof, orthologs thereof, or modified versions thereof. Insome embodiments, the Cas protein has DNA or RNA cleavage activity. Insome embodiments, the Cas protein directs cleavage of one or bothstrands of a nucleic acid molecule at the location of a target sequence,such as within the target sequence and/or within the complement of thetarget sequence. In some embodiments, the Cas protein directs cleavageof one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 50, 100, 200, 500, or more base pairs from the first or lastnucleotide of a target sequence.

In one embodiment, the CRISPR-Cas domain is associated with one or morefunctional domains. For example, in one embodiment, one or morefunctional domains may be associated with (i.e. bound to or fused with)the C terminus or the N terminus of the Cas9 enzyme or homolog orortholog thereof. In such an embodiment, the functional domains aretypically fused via a linker. In another embodiment, one or morefunctional domains may be provided along with the Cas9 enzyme or homologor ortholog thereof.

The one or more functional domains may have one or more activitiesincluding, but not limited to, cytidine deaminase activity, methylaseactivity, demethylase activity, transcription activation activity,transcription repression activity, transcription release factoractivity, histone modification activity, RNA cleavage activity, DNAcleavage activity, DNA integration activity or nucleic acid bindingactivity. The one or more functional domains may be transcriptionalactivation domain or a repressor domains.

Activator and repressor domains which may further modulate function maybe selected on the basis of species, strength, mechanism, duration,size, or any number of other parameters. Exemplary effector domainsinclude, but are not limited to, a transposase domain, integrase domain,recombinase domain, resolvase domain, invertase domain, protease domain,DNA methyltransferase domain, DNA demethylase domain, histone acetylasedomain, histone deacetylases domain, nuclease domain, repressor domain,activator domain, nuclear-localization signal domains,transcription-protein recruiting domain, cellular uptake activityassociated domain, nucleic acid binding domain or antibody presentationdomain.

In one embodiment, the one or more functional domain may be an APOBECdomain (see e.g., Yang et al., J Genet Genomics. 44(9):423-437 (2017)).APOBEC-catalyzed deamination in single-stranded nucleic acid moleculescan be further processed to yield mutations including, but not limitedto, insertions or deletions (indels).

Polypeptide Inhibitors

In some embodiments, the invention includes an isolated peptideinhibitor that inhibits group II intron splicing. For example, in oneembodiment, the peptide inhibitor of the invention inhibits group IIintron splicing directly by binding to a 3-dimensional structure formedduring group II intron folding thereby preventing the self-splicingactivity of the group II intron.

The variants of the polypeptides according to the present invention maybe (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue andsuch substituted amino acid residue may or may not be one encoded by thegenetic code, (ii) one in which there are one or more modified aminoacid residues, e.g., residues that are modified by the attachment ofsubstituent groups, (iii) one in which the polypeptide is an alternativesplice variant of the polypeptide of the present invention, (iv)fragments of the polypeptides and/or (v) one in which the polypeptide isfused with another polypeptide, such as a leader or secretory sequenceor a sequence which is employed for purification (for example, His-tag)or for detection (for example, Sv5 epitope tag). The fragments includepolypeptides generated via proteolytic cleavage (including multi-siteproteolysis) of an original sequence. Variants may bepost-translationally, or chemically modified. Such variants are deemedto be within the scope of those skilled in the art from the teachingherein.

Combinations

In one embodiment, the composition of the present invention comprises acombination of an inhibitor of group II intron splicing and secondtherapeutic agent. For example, in various embodiments the secondtherapeutic agent includes, but is not limited to an antifungal agent,an antibacterial agent, a parasite infection therapeutic and a yeastinfection therapeutic.

In some embodiments, a composition comprising a combination ofinhibitors described herein has an additive effect, wherein the overalleffect of the combination is approximately equal to the sum of theeffects of each individual inhibitor. In other embodiments, acomposition comprising a combination of inhibitors described herein hasa synergistic effect, wherein the overall effect of the combination isgreater than the sum of the effects of each individual inhibitor.

A composition comprising a combination of inhibitors comprisesindividual inhibitors in any suitable ratio. For example, in oneembodiment, the composition comprises a 1:1 ratio of two individualinhibitors. However, the combination is not limited to any particularratio. Rather any ratio that is shown to be effective is encompassed.

Therapeutic Methods

The present invention also provides methods of treating or preventing adisease or disorder associated with an organism that harbors a group IIintron in a subject. In one embodiment, the disease or disorder is abacterial, parasite, yeast or fungal infection. In one embodiment, thedisease or disorder is associated with a bacterial, parasite, yeast orfungal infection.

It will be appreciated by one of skill in the art, when armed with thepresent disclosure including the methods detailed herein, that theinvention is not limited to treatment of a disease or disorder that isalready established. Particularly, the disease or disorder need not havemanifested to the point of detriment to the subject; indeed, the diseaseor disorder need not be detected in a subject before treatment isadministered. That is, significant signs or symptoms of a disease ordisorder do not have to occur before the present invention may providebenefit. Therefore, the present invention includes a method forpreventing a disease or disorder, in that a composition, as discussedpreviously elsewhere herein, can be administered to a subject prior tothe onset of a disease or disorder, thereby preventing a disease ordisorder.

Exemplary bacteria harboring group II introns include, but are notlimited to, Azotobacter vinelandii; Bacillus anthracis; Bacillus cereus;Bacillus halorudans; Bacillus megaterium; Calothrix species; Clostridiumdifficile; Escherichia coli; Lactococcus lactis; Legionella pneumophila;Pseudomonas alcaligenes; Pseudomonas putida; Pseudomonas sp.; Serratiamarcescens; Sinorhizobium meliloti; Sphingomonas aromaticivorans;Shigella flexneri; and Streptococcus pneumoniae.

Exemplary yeast harboring group II introns include, but are not limitedto, Candida parapsilosis; Canida zemplinina; Candida Ipomoeae;Saccharomyces cerevisiae; and Schizosaccharomyces pombe.

Exemplary fungi harboring group II introns include, but are not limitedto, Ceripopriopsis subvermispora; Coccidioides immitis; Cordycepskonnoana; Cryphonectria parasitica; Glomus intraradices;Paracoccidioides brasiliensis; Podospora anserine; Tremetes cingulate;and Usnea Antarctica.

One of skill in the art, when armed with the disclosure herein, wouldappreciate that the prevention of a bacterial, parasite, yeast or fungalinfection, encompasses administering to a subject a composition as apreventative measure against the development of, or progression of abacterial, parasite, yeast or fungal infection.

The invention encompasses administration of an inhibitor of group IIintron splicing. To practice the methods of the invention; the skilledartisan would understand, based on the disclosure provided herein, howto formulate and administer the appropriate inhibitor composition to asubject. The present invention is not limited to any particular methodof administration or treatment regimen.

In one embodiment, the method comprises administering to the subject inneed an effective amount of a composition that reduces or inhibits theself-splicing activity of a group II intron.

One of skill in the art will appreciate that the inhibitors of theinvention can be administered singly or in any combination. Further, theinhibitors of the invention can be administered singly or in anycombination in a temporal sense, in that they may be administeredconcurrently, or before, and/or after each other. One of ordinary skillin the art will appreciate, based on the disclosure provided herein,that the inhibitor compositions of the invention can be used to preventor to treat a bacterial, parasite, yeast or fungal infection, and thatan inhibitor composition can be used alone or in any combination withanother modulator to effect a therapeutic result. In variousembodiments, any of the inhibitor compositions of the inventiondescribed herein can be administered alone or in combination with othermodulators of other molecules associated with a bacterial, parasite,yeast or fungal infection.

In one embodiment, the invention includes a method comprisingadministering a combination of inhibitors described herein. In someembodiments, the method has an additive effect, wherein the overalleffect of the administering a combination of inhibitors is approximatelyequal to the sum of the effects of administering each individualinhibitor. In other embodiments, the method has a synergistic effect,wherein the overall effect of administering a combination of inhibitorsis greater than the sum of the effects of administering each individualinhibitor.

The method comprises administering a combination of inhibitors in anysuitable ratio. For example, in one embodiment, the method comprisesadministering two individual inhibitors at a 1:1 ratio. However, themethod is not limited to any particular ratio. Rather any ratio that isshown to be effective is encompassed.

Gene Therapy

As generally discussed above, a nucleic acid, where applicable, may beemployed in gene therapy methods in order to inhibit group II intronsplicing. “Gene therapy” includes both conventional gene therapy where alasting effect is achieved by a single treatment, and the administrationof gene therapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA.Oligonucleotides can be modified to enhance their uptake, e.g., bysubstituting their negatively charged phosphodiester groups by unchargedgroups. One or more vector encoding an inhibitor of group II intronsplicing of the present invention can be delivered using gene therapymethods, for example locally in a cell or tissue or systemically (e.g.,via vectors that selectively target specific tissue types, for example,tissue-specific adeno-associated viral vectors). In some embodiments,primary cells harvested from an individual can be transfected ex vivowith a nucleic acid encoding an inhibitor of group II intron splicing ofthe present invention, and then returned the transfected cells to theindividual's body.

In some aspects, the invention provides methods comprising deliveringone or more polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and organisms (such asanimals, plants, or fungi) comprising or produced from such cells. Insome embodiments, a CRISPR enzyme in combination with (and optionallycomplexed with) a guide sequence is delivered to a cell. Conventionalviral and non-viral based gene transfer methods can be used to introducenucleic acids in cells or target tissues. Such methods can be used toadminister nucleic acids encoding an inhibitor of group II intronsplicing (e.g., encoding components of a CRISPR system) to cells inculture, or in a host organism. Non-viral vector delivery systemsinclude DNA plasmids, RNA (e.g. a transcript of a vector describedherein), naked nucleic acid, and nucleic acid complexed with a deliveryvehicle, such as a liposome. For a review of gene therapy procedures,see e.g., Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):31-44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology Doerfler and Bohm (eds) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

Other Applications

In one embodiment, the group II splicing inhibitors of the invention areuseful for the inhibition, treatment or prevention of growth of anorganism harboring an active group II intron. Although the inventionfinds utility as a therapeutic agent, the present invention is notlimited to the treatment or prevention of growth of an organismharboring an active group II intron in the body or in medical settings.It is known in the art that organisms harboring an active group IIintron can grow on a variety of surfaces which can lead to diversedetrimental issues. For example, organism growth in kitchen or bathroomsurfaces may present a host of sanitation issues. In an alternativeembodiment, organism growth on physical surfaces and substrates,engineering systems or vehicles may lead to corrosion and biofouling.Thus, the present invention encompasses the inhibition, treatment orprevention of growth of organisms harboring group II introns that mayoccur in environmental surfaces and substrates, residential surfaces andsubstrates, commercial surfaces and substrates, and industrial surfacesand substrates. Therefore, the present invention encompasses theinhibition, treatment or prevention of growth of organisms harboringgroup II introns that may occur in environmental, residential,commercial, industrial, or other settings.

In one embodiment, growth of an organism that harbors a group II intronresults in the formation of a biofilm. Therefore, in one embodiment, thepresent invention provides a composition that inhibits the formation ofbiofilms. In one embodiment, the composition inhibits the accumulationof biofilm. In one embodiment, the composition promotes the disruptionor disassembly of an existing biofilm.

The present invention is not limited to treating and/or preventingbiofilms in a living body, but rather encompasses methods of treatingand/or preventing biofilms on surfaces outside the body (e.g., includingenvironmental surfaces and substrates, commercial surfaces andsubstrates, residential surfaces and substrates, and industrial surfacesand substrates, etc.). For example, biofilms can form on surfaces indamp environments including bathrooms, kitchens, and certainresidential, commercial and industrial settings. In some embodiments,the composition described herein is used in a method to treat and/orprevent biofilm formation or accumulation along surfaces in residential,commercial, military, maritime, and industrial settings. The methodcomprises administering an effective amount of an inhibitor of group IIintron splicing to the surface, or to incorporating an effective amountof an inhibitor of group II intro splicing into a surface, substrate, orother protective surface or material.

In one embodiment, the inhibitor of group II intron splicing isincorporated into a marine coating, such as a marine coating comprisinga film forming agent and solvent and optionally other ingredients. Thesolvent may be either organic solvent or water. The inhibitors of groupII intron splicing of the invention are suitable for use in both solventand water based marine coatings. Any conventional film forming agent maybe utilized in the marine coating incorporating the inhibitors of theinvention. Film-forming components may include polymer resin solutions.Non-limiting examples of polymer resins include unsaturated polyesterresins formed from (a) unsaturated acids and anhydrides, such as maleicanhydride, fumaric acid, and itaconic acid; (b) saturated acids andanhydrides, such as phthalic anhydride, isophthalic anhydride,terephthalic anhydride, tetrahydrophthalic anhydride, tetrahalophthalicanhydrides, chlorendic acid, adipic acid, and sebacic acid; (c) glycols,such as ethylene glycol, 1,2 propylene glycol, dibromoneopentyl glycol,Dianol 33®, and Dianol 22®; and (d) vinyl monomers, such as styrene,vinyl toluene, chlorostyrene, bromostyrene, methylmethacrylate, andethylene glycol dimethacrylate. Other suitable resins include, but arenot limited to, vinyl ester-, vinyl acetate-, and vinyl chloride-basedresins, vinyl chloride-vinyl acetate copolymer systems as aqueousdispersions or solvent based systems, mixtures of natural rosin andvinyl chloride-vinyl acetate copolymers, acrylic resins in solvent basedor aqueous systems, urethane-based resins, self-polishing copolymerresins, ablative resins, leaching resins, elastomeric components,vulcanized rubbers, butadiene-styrene rubbers, butadiene-acrylonitrilerubbers, butadiene-styrene-acrylonitrile rubbers, drying oils such aslinseed oil, asphalt, epoxies, siloxanes like for examplepolydimethylsiloxane, silanes like alkyl and aryl alkoxy silanes,silicones and silicone-based technologies like fluorosilicones, siliconeacrylates, silicone latex elastomers, and combinations thereof.

The marine coating composition of the invention may include componentsin addition to inhibitors of group II intron splicing and a film-formingcomponent, so as to confer one or more desirable properties, such asincreased or decreased hardness, strength, increased or decreasedrigidity, reduced drag, increased or decreased permeability, or improvedwater resistance. The selection of a particular component or group ofcomponents to impart such properties are within the capabilities ofthose having ordinary skill in the art. The marine coatings of thepresent invention may optionally contain one or more of the following:inorganic pigments, organic pigments or dyes, natural resins, such asrosin, fillers, extenders, swelling agents, wetting agents, coalescents,plasticizers, dispersants, surface active agents, preservatives,rheology modifiers or adhesion promoters, UV filters, and combinationsthereof.

In some embodiments, the composition of the invention comprises a paint,sealant, solution, wax, spray, or the like, which comprises one or moreinhibitors of group II intron splicing as described above.

The primary active ingredient for use in the present invention comprisesat least inhibitor of group II intron splicing as described above. Thepercentage of inhibitor in the coating composition required foreffective protection against biofouling organisms may vary substantiallydepending on the nature of the marine or freshwater structure to whichthe coating composition is applied, the service in which the structureis used, the pH of water and other environmental conditions to which thestructure is exposed, depending on the inhibitor itself, the chemicalnature of the film former, as well as other additives present in thecomposition that may influence the effectiveness of the inhibitor ofgroup II intron splicing. The upper limit of activity may be also drivenby characteristics of cost and toxicity that would be readily apparentto the skilled artisan. One skilled in the art would recognize that theamount of group II intron inhibitor could be reduced in the event asecond active ingredient were present, so long as the combinedcomposition is active as an antifoulant.

Also within the scope of this invention is any article, substrate,surface, or material having incorporated or having a surface coated,with a composition containing at least one inhibitor of group II intronsplicing or derivative or salt or a combination thereof. The impregnatedand/or coated articles of the invention can comprise any material thatis in contact with fresh, salt, estuarine, brackish, sea or other bodiesof water to which biofouling organisms are prone to attach, such asmetal, wood, concrete, plastic, composite, and stone. Representativeexamples of articles which may benefit from a coating which inhibitsattachment and growth of such organisms include boats and ships, forexample their hulls, propellers, rudders, keels, centerboards, fins,hydrofoils, berthing facilities, such as piers and pilings, decksurfaces, buoys, wharves, jetties, fishing nets, industrial coolingsystem surfaces, cooling water intake, or discharge pipes,desalinization facilities, nautical beacons, floating beacons, floatingbreakwaters, docks, pipes, pipelines, tanks, water pipes in powerstations, seaside industrial plants, fish preserving structures, aquaticconstructions, port facilities, bridges, bells, plumbs, wheels, cranes,dredges, pumps, valves, wires, cables, ropes, ladders, pontoons,transponders, antennae, barges, periscopes, snorkels, gun mounts, gunbarrels, launch tubes, mines, offshore rigging equipment, intake screensfor water distribution systems and decorative or functional cement orstone formations.

In some embodiments, the composition described herein is used in amethod to inhibit, treat and/or prevent growth of fungi, plants, algae,Euglena or other organisms that harbor an active group II intron. Themethod comprises administering an effective amount of an inhibitor ofgroup II intron splicing to the organism.

Exemplary fungi identified as potential maritime biofouling pestsinclude, but are not limited to, Aspergillus niger, Aspergillus sp.,Aspergillus versicolor, Fusarium solani, Paecilomyces lilacinus,Paecilomyces nivea, Paecilomyces spectabilis, Penicillium chrysogenum,Penicillium sp., and Trichoderma sp.

Exemplary algae identified as potential maritime biofouling pestsinclude, but are not limited to, members of the Divisions Chlorophyta(green algae), Chrysophyta (yeliow-green algae), Cyanophyta (blue-greenalgae or bacteria), Euglenophyta (euglenoides), Phaeophyta (brownalgae), Xanthophyta (yellow-green algae), Pyrrophyta (fire algae), andRhodophyta (red algae). Specific examples of algae include, but are notlimited to, Anabaena, Botryococcus braunii, Chlamydomonas reinhardii,Chlorella sp., Crypthecodinium cohnii, Cylindrotheca sp., Didymospheniageminata, Dunaliella primolecta, Dunaliella tertiolecta, Euglenagracilis, Gracilaria Salicornia, Hydrodictyon reticulatum, Isochrysisgalbana, Kappaphycus/Eucheuma, Lyngbya, Monallanthus salina,Nannochloris sp., Nannochloropsis salina, Neochloris oleoabundans,Nitzschia sp., Oscillatoria, Phaeodactylum tricornutum, Pithophora spp.,Pleurochrysis carterae, Pylaiella littoralis, Scenedesmus dimorphus,Schizochytrium sp; Spirogyra spp, Spirulina sp., Tetraselmis chuff, andTetraselmis suecica.

Exemplary plants harboring group II introns include, but are not limitedto, Citrullus lanatus, Chaetosphaeridium globosum, Chlorokybusatmophyticus, Nicotiana tabacum, Marchantia polymorph, Oenothera,Pedinomonas minor, Spirodela oligorhiz, Zea mays, Hordeum vulgare,Sinapis alba, lathyrus sativus, Scenedesmus obliquus, Spinacia oleracea,and Triticum aestivum. Further examples of pest water weeds include, butare not limited to, Egeria densa and Ceratophyllum demersum.

Pharmaceutical Compositions and Formulations

The invention also encompasses the use of pharmaceutical compositions ofthe invention or salts thereof to practice the methods of the invention.Such a pharmaceutical composition may consist of at least one inhibitorcomposition of the invention or a salt thereof in a form suitable foradministration to a subject, or the pharmaceutical composition maycomprise at least one inhibitor composition of the invention or a saltthereof, and one or more pharmaceutically acceptable carriers, one ormore additional ingredients, or some combination of these. The compoundor conjugate of the invention may be present in the pharmaceuticalcomposition in the form of a physiologically acceptable salt, such as incombination with a physiologically acceptable cation or anion, as iswell known in the art.

In an embodiment, the pharmaceutical compositions useful for practicingthe methods of the invention may be administered to deliver a dose ofbetween 1 ng/kg/day and 100 mg/kg/day. In another embodiment, thepharmaceutical compositions useful for practicing the invention may beadministered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient. In various embodiments, the composition comprises about 1%,about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%,about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%,about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%,about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%,about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%,about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%,about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,about 100% (w/w) active ingredient (e.g., inhibitor, etc.).

Pharmaceutical compositions that are useful in the methods of theinvention may be suitably developed for oral, rectal, vaginal,parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, oranother route of administration. A composition useful within the methodsof the invention may be directly administered to the skin, vagina or anyother tissue of a mammal. Other contemplated formulations includeliposomal preparations, resealed erythrocytes containing the activeingredient, and immunologically-based formulations. The route(s) ofadministration will be readily apparent to the skilled artisan and willdepend upon any number of factors including the type and severity of thedisease being treated, the type and age of the veterinary or humansubject being treated, and the like.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

As used herein, a “unit dose” is a discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient that would be administered to a subject or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage. The unit dosage form may be for a singledaily dose or one of multiple daily doses (e.g., about 1 to 4 or moretimes per day). When multiple daily doses are used, the unit dosage formmay be the same or different for each dose.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions that aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist maydesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

In one embodiment, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Inone embodiment, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a compound or conjugateof the invention and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers that are useful, include, but arenot limited to, glycerol, water, saline, ethanol and otherpharmaceutically acceptable salt solutions such as phosphates and saltsof organic acids. Examples of these and other pharmaceuticallyacceptable carriers are described in Remington's Pharmaceutical Sciences(1991, Mack Publication Co., New Jersey).

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, isotonic agents will be included, for example, sugars, sodiumchloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of the injectable compositions may bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate or gelatin. In oneembodiment, the pharmaceutically acceptable carrier is not DMSO alone.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, vaginal, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” that may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed. (1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, PA), which isincorporated herein by reference.

The composition of the invention may comprise a preservative from about0.005% to 2.0% by total weight of the composition. The preservative isused to prevent spoilage in the case of exposure to contaminants in theenvironment. Examples of preservatives useful in accordance with theinvention included but are not limited to those selected from the groupconsisting of benzyl alcohol, sorbic acid, parabens, imidurea andcombinations thereof. An exemplary preservative is a combination ofabout 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

In one embodiment, the composition includes an anti-oxidant and achelating agent that inhibits the degradation of the compound.Antioxidants for some compounds include, but are not limited to, BHT,BHA, alpha-tocopherol and ascorbic acid in the range of about 0.01% to0.3% (e.g., BHT in the range of 0.03% to 0.1% by weight by total weightof the composition). In one embodiment, the chelating agent is presentin an amount of from 0.01% to 0.5% by weight by total weight of thecomposition. Chelating agents include, but are not limited to, edetatesalts (e.g. disodium edetate) and citric acid in the weight range ofabout 0.01% to 0.20% by weight by total weight of the composition. Thechelating agent is useful for chelating metal ions in the compositionthat may be detrimental to the shelf life of the formulation. While BHTand disodium edetate are an exemplary antioxidant and chelating agentrespectively for some compounds, other suitable and equivalentantioxidants and chelating agents may be substituted therefore as wouldbe known to those skilled in the art.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water, and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.,polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin, and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. As used herein, an “oily” liquidis one which comprises a carbon-containing liquid molecule and whichexhibits a less polar character than water. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water, and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the subjecteither prior to or after a diagnosis of disease. Further, severaldivided dosages, as well as staggered dosages may be administered dailyor sequentially, or the dose may be continuously infused, or may be abolus injection. Further, the dosages of the therapeutic formulationsmay be proportionally increased or decreased as indicated by theexigencies of the therapeutic or prophylactic situation.

Administration of the compositions of the present invention to asubject, (e.g., a mammal, including but not limited to, a human) may becarried out using known procedures, at dosages and for periods of timeeffective to prevent or treat disease. An effective amount of thetherapeutic compound necessary to achieve a therapeutic effect may varyaccording to factors such as the activity of the particular compoundemployed; the time of administration; the rate of excretion of thecompound; the duration of the treatment; other drugs, compounds ormaterials used in combination with the compound; the state of thedisease or disorder, age, sex, weight, condition, general health andprior medical history of the subject being treated, and like factorswell-known in the medical arts. Dosage regimens may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily, or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation. Anon-limiting example of an effective dose range for a therapeuticcompound of the invention is from about 1 and 5,000 mg/kg of bodyweight/per day. One of ordinary skill in the art would be able to studythe relevant factors and make the determination regarding the effectiveamount of the therapeutic compound without undue experimentation.

The compound may be administered to a subject as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on. The frequency of the dose will bereadily apparent to the skilled artisan and will depend upon any numberof factors, such as, but not limited to, the type and severity of thedisease being treated, the type and age of the animal, etc.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular subject, composition, and mode ofadministration, without being toxic to the subject.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of a disease in a subject.

In one embodiment, the compositions of the invention are administered tothe subject in dosages that range from one to five times per day ormore. In another embodiment, the compositions of the invention areadministered to the subject in range of dosages that include, but arenot limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It will be readily apparent toone skilled in the art that the frequency of administration of thevarious combination compositions of the invention will vary from subjectto subject depending on many factors including, but not limited to, age,disease or disorder to be treated, gender, overall health, and otherfactors. Thus, the invention should not be construed to be limited toany particular dosage regime and the precise dosage and composition tobe administered to any subject will be determined by the attendingphysical taking all other factors about the subject into account.

Compounds of the invention for administration may be in the range offrom about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg toabout 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg toabout 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about400 mg to about 500 mg, and any and all whole or partial incrementsthere between.

In some embodiments, the dose of a compound of the invention is fromabout 1 mg and about 2,500 mg. In some embodiments, a dose of a compoundof the invention used in compositions described herein is less thanabout 10,000 mg, or less than about 8,000 mg, or less than about 6,000mg, or less than about 5,000 mg, or less than about 3,000 mg, or lessthan about 2,000 mg, or less than about 1,000 mg, or less than about 500mg, or less than about 200 mg, or less than about 50 mg. Similarly, insome embodiments, a dose of a second compound (i.e., a drug used fortreating the same or another disease as that treated by the compositionsof the invention) as described herein is less than about 1,000 mg, orless than about 800 mg, or less than about 600 mg, or less than about500 mg, or less than about 400 mg, or less than about 300 mg, or lessthan about 200 mg, or less than about 100 mg, or less than about 50 mg,or less than about 40 mg, or less than about 30 mg, or less than about25 mg, or less than about 20 mg, or less than about 15 mg, or less thanabout 10 mg, or less than about 5 mg, or less than about 2 mg, or lessthan about 1 mg, or less than about 0.5 mg, and any and all whole orpartial increments thereof.

In one embodiment, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound or conjugate of theinvention, alone or in combination with a second pharmaceutical agent;and instructions for using the compound or conjugate to treat, prevent,or reduce one or more symptoms of a disease in a subject.

The term “container” includes any receptacle for holding thepharmaceutical composition. For example, in one embodiment, thecontainer is the packaging that contains the pharmaceutical composition.In other embodiments, the container is not the packaging that containsthe pharmaceutical composition, i.e., the container is a receptacle,such as a box or vial that contains the packaged pharmaceuticalcomposition or unpackaged pharmaceutical composition and theinstructions for use of the pharmaceutical composition. Moreover,packaging techniques are well known in the art. It should be understoodthat the instructions for use of the pharmaceutical composition may becontained on the packaging containing the pharmaceutical composition,and as such the instructions form an increased functional relationshipto the packaged product. However, it should be understood that theinstructions may contain information pertaining to the compound'sability to perform its intended function, e.g., treating or preventing adisease in a subject, or delivering an imaging or diagnostic agent to asubject.

Routes of administration of any of the compositions of the inventioninclude oral, nasal, rectal, parenteral, sublingual, transdermal,transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral,vaginal (e.g., trans- and perivaginally), (intra)nasal, and(trans)rectal), intravesical, intrapulmonary, intraduodenal,intragastrical, intrathecal, subcutaneous, intramuscular, intradermal,intra-arterial, intravenous, intrabronchial, inhalation, and topicaladministration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Otherformulations suitable for oral administration include, but are notlimited to, a powdered or granular formulation, an aqueous or oilysuspension, an aqueous or oily solution, a paste, a gel, toothpaste, amouthwash, a coating, an oral rinse, chewing gum, varnishes, sealants,oral and teeth “dissolving strips”, or an emulsion. The compositionsintended for oral use may be prepared according to any method known inthe art and such compositions may contain one or more agents selectedfrom the group consisting of inert, non-toxic pharmaceuticallyexcipients that are suitable for the manufacture of tablets. Suchexcipients include, for example an inert diluent such as lactose;granulating and disintegrating agents such as cornstarch; binding agentssuch as starch; and lubricating agents such as magnesium stearate.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and U.S. Pat. No. 4,265,874 to formosmotically controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide for pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

For oral administration, the compositions of the invention may be in theform of tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents; fillers;lubricants; disintegrates; or wetting agents. If desired, the tabletsmay be coated using suitable methods and coating materials such asOPADRY™ film coating systems available from Colorcon, West Point, Pa.(e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, AqueousEnteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400).

Liquid preparation for oral administration may be in the form ofsolutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid). Liquid formulations of a pharmaceuticalcomposition of the invention which are suitable for oral administrationmay be prepared, packaged, and sold either in liquid form or in the formof a dry product intended for reconstitution with water or anothersuitable vehicle prior to use.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface-active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Granulating techniques are well known in the pharmaceutical art formodifying starting powders or other particulate materials of an activeingredient. The powders are typically mixed with a binder material intolarger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using “wet” granulation processesare generally characterized in that the powders are combined with abinder material and moistened with water or an organic solvent underconditions resulting in the formation of a wet granulated mass fromwhich the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that aresolid or semi-solid at room temperature (i.e. having a relatively lowsoftening or melting point range) to promote granulation of powdered orother materials, essentially in the absence of added water or otherliquid solvents. The low melting solids, when heated to a temperature inthe melting point range, liquefy to act as a binder or granulatingmedium. The liquefied solid spreads itself over the surface of powderedmaterials with which it is contacted, and on cooling, forms a solidgranulated mass in which the initial materials are bound together. Theresulting melt granulation may then be provided to a tablet press or beencapsulated for preparing the oral dosage form. Melt granulationimproves the dissolution rate and bioavailability of an active (i.e.drug) by forming a solid dispersion or solid solution.

U.S. Pat. No. 5,169,645 discloses directly compressible wax-containinggranules having improved flow properties. The granules are obtained whenwaxes are admixed in the melt with certain flow improving additives,followed by cooling and granulation of the admixture. In someembodiments, only the wax itself melts in the melt combination of thewax(es) and additives(s), and in other cases both the wax(es) and theadditives(s) will melt.

The present invention also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compounds of theinvention, and a further layer providing for the immediate release of amedication for treatment of a disease. Using a wax/pH-sensitive polymermix, a gastric insoluble composition may be obtained in which the activeingredient is entrapped, ensuring its delayed release.

Parenteral Administration

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen-free water) prior toparenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulations thatare useful include those that comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer system. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Topical Administration

An obstacle for topical administration of pharmaceuticals is the stratumcorneum layer of the epidermis. The stratum corneum is a highlyresistant layer comprised of protein, cholesterol, sphingolipids, freefatty acids and various other lipids, and includes cornified and livingcells. One of the factors that limit the penetration rate (flux) of acompound through the stratum corneum is the amount of the activesubstance that can be loaded or applied onto the skin surface. Thegreater the amount of active substance which is applied per unit of areaof the skin, the greater the concentration gradient between the skinsurface and the lower layers of the skin, and in turn the greater thediffusion force of the active substance through the skin. Therefore, aformulation containing a greater concentration of the active substanceis more likely to result in penetration of the active substance throughthe skin, and more of it, and at a more consistent rate, than aformulation having a lesser concentration, all other things being equal.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions. Topicallyadministrable formulations may, for example, comprise from about 1% toabout 10% (w/w) active ingredient, although the concentration of theactive ingredient may be as high as the solubility limit of the activeingredient in the solvent. Formulations for topical administration mayfurther comprise one or more of the additional ingredients describedherein.

Enhancers of permeation may be used. These materials increase the rateof penetration of drugs across the skin. Typical enhancers in the artinclude ethanol, glycerol monolaurate, PGML (polyethylene glycolmonolaurate), dimethylsulfoxide, and the like. Other enhancers includeoleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylicacids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone.

One acceptable vehicle for topical delivery of some of the compositionsof the invention may contain liposomes. The composition of the liposomesand their use are known in the art (for example, see U.S. Pat. No.6,323,219).

In alternative embodiments, the topically active pharmaceuticalcomposition may be optionally combined with other ingredients such asadjuvants, anti-oxidants, chelating agents, surfactants, foaming agents,wetting agents, emulsifying agents, viscosifiers, buffering agents,preservatives, and the like. In another embodiment, a permeation orpenetration enhancer is included in the composition and is effective inimproving the percutaneous penetration of the active ingredient into andthrough the stratum corneum with respect to a composition lacking thepermeation enhancer. Various permeation enhancers, including oleic acid,oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids,dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone, are known tothose of skill in the art. In another embodiment, the composition mayfurther comprise a hydrotropic agent, which functions to increasedisorder in the structure of the stratum corneum, and thus allowsincreased transport across the stratum corneum. Various hydrotropicagents, such as isopropyl alcohol, propylene glycol, or sodium xylenesulfonate, are known to those of skill in the art.

The topically active pharmaceutical composition should be applied in anamount effective to affect desired changes. As used herein “amounteffective” shall mean an amount sufficient to cover the region of skinsurface where a change is desired. An active compound should be presentin the amount of from about 0.0001% to about 15% by weight volume of thecomposition. In one embodiment, it should be present in an amount fromabout 0.0005% to about 5% of the composition. In one embodiment, itshould be present in an amount of from about 0.001% to about 1% of thecomposition. Such compounds may be synthetically- or naturally derived.

Rectal Administration

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non-irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e., about 20° C.) and which isliquid at the rectal temperature of the subject (i.e., about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants, andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants, and preservatives.

Vaginal Administration

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for vaginal administration. Withrespect to the vaginal or perivaginal administration of the compounds ofthe invention, dosage forms may include vaginal suppositories, creams,ointments, liquid formulations, pessaries, tampons, gels, pastes, foamsor sprays. The suppository, solution, cream, ointment, liquidformulation, pessary, tampon, gel, paste, foam or spray for vaginal orperivaginal delivery comprises a therapeutically effective amount of theselected active agent and one or more conventional nontoxic carrierssuitable for vaginal or perivaginal drug administration. The vaginal orperivaginal forms of the present invention may be manufactured usingconventional processes as disclosed in Remington: The Science andPractice of Pharmacy, supra (see also drug formulations as adapted inU.S. Pat. Nos. 6,515,198; 6,500,822; 6,417,186; 6,416,779; 6,376,500;6,355,641; 6,258,819; 6,172,062; and 6,086,909). The vaginal orperivaginal dosage unit may be fabricated to disintegrate rapidly orover a period of several hours. The time period for completedisintegration may be in the range of from about 10 minutes to about 6hours, e.g., less than about 3 hours.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made bycombining the active ingredient with a pharmaceutically acceptableliquid carrier. As is well known in the art, douche preparations may beadministered using, and may be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject.

Douche preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants, antibiotics, antifungalagents, and preservatives.

Buccal Administration

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed, mayhave an average particle or droplet size in the range from about 0.1 toabout 200 nanometers, and may further comprise one or more of theadditional ingredients described herein. The examples of formulationsdescribed herein are not exhaustive and it is understood that theinvention includes additional modifications of these and otherformulations not described herein, but which are known to those of skillin the art.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389,5,582,837, and 5,007,790. Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Applications Nos.20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and20020051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041, WO03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO98/11879, WO 97/47285, WO 93/18755, and WO 90/11757.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the exemplary embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1: Small Molecules that Target Group II Intron RNA are PotentAntifungal Agents

In this study, compound libraries were screened for small molecules thatspecifically inhibit the splicing of group II introns from yeast, andtheir activities were monitored in-vitro and in-vivo. The most promisingscaffold compounds were optimized to yield a set of high affinity groupII intron splicing inhibitors that are potent against the pathogenicyeast C. parapsilosis, and which are not toxic in mammalian cells. Giventhe unique RNA metabolism of fungi and yeast, these results demonstratethat RNA targeting may provide a much-needed approach for developingtherapeutics against eukaryotic pathogens. These results alsodemonstrate that RNA tertiary structures can be targeted de-novo andthat high affinity compounds can be identified using classical methodsfor developing pharmacologically active compounds. Given the vast numberof physiologically important RNA tertiary structures that control geneexpression in all domains of life, these results suggest a pathway fortargeting these RNAs, resulting in a new generation of small moleculetherapeutics.

The materials and methods employed in these experiments are nowdescribed.

Yeast Strains.

Strains of Candida parapsilosis (ATCC 22019) and Saccharomycescerevisiae (ATCC 18824) were purchased from American Type CultureCollection (ATCC) and cultured according to the instructions from themanufacturer (ATCC). S. cerevisiae Wild-type (NP40-36a) and mtDNAintronless (XPM46) strains were provided (Perez-Martinez et al., 2003,EMBO J 22:5951-5961).

RNA Preparation.

Synthesis of the RNA Oligo Substrates.

RNA oligonucleotides containing 3′-terminal Black Hole Quencher 2 labeland aminomodifier C6dT nucleotide (Glen Research) as well as U2-U6 RNAoligonucleotide (AGCAGUUCCCCUGCAUAAGGAUGAACCGCU (SEQ ID NO:19) weresynthesized on a MerMade 12 DNA-RNA synthesizer (BioAutomation) usingTBDMS phosphoramidites (Glen Research). Base deprotection was carriedout in 3:1 mixture of 30% ammonium hydroxide (JT Baker) and ethanol atroom temperature for 24 hours. Subsequent 2′-OH deprotection andpurification on a 20% denaturing polyacrylamide gel were carried out aspreviously described (Wincott et al., 1995, Nucleic acids research,23:2677-2684; Pyle and Green, 1994, Biochemistry, 33:2716-2725). RNAoligonucleotide U2-U6 was deprotected and purified as described (Wincottet al., 1995, Nucleic acids research, 23:2677-2684; Pyle and Green,1994, Biochemistry, 33:2716-2725).

In Vitro Transcription.

Large-scale transcription of the SE, D1(Domain 1 in isolation), D3(Domain 3 in isolation), D56 (RNA molecule containing both domains 5 and6) and D135 RNAs (Su et al., 2005, Nucleic acids research, 33:6674-6687;Swisher et al., 2001, EMBO J, 20:2051-2061; Swisher et al., 2002,Journal of molecular biology, 315:297-310) was carried out using T7 RNApolymerase as previously described (Pyle and Green, 1994, Biochemistry,33:2716-2725). Internally ³²P-labeled ai5γ intron with short exons (SERNA) (Zingler et al., 2010, Nucleic acids research, 38:6602-6609) wasprepared by in vitro transcription as described (Pyle and Green, 1994,Biochemistry, 33:2716-2725; Daniels et al., 1996, Journal of molecularbiology, 256:31-49). Internally labeled Azo-Pre-tRNA (Tanner et al.,1996, RNA 2:74-83) was in vitro transcribed and purified as previouslydescribed (Tanner et al., 1996, RNA 2:74-83). RNA molecules D3 and D56were purified on an 8% denaturing polyacrylamide gel, and all other RNAswere purified on a 5% gel as previously described (Pyle and Green, 1994,Biochemistry, 33:2716-2725; Daniels et al., 1996, Journal of molecularbiology, 256:31-49).

Fluorescent Labeling of RNA Oligonucleotides

Purified RNA oligonucleotides containing 3′-terminal Black Hole Quencher2 label and aminomodifier C6dT nucleotide (Glen Research) werefluorescently labeled with the NHS ester of AlexaFluor 555 dye (LifeTechnologies Corp.) at the primary amino group located on theaminomodifier C6dT nucleotide. RNA oligonucleotides were dissolved in200 μl of 0.25 M sodium bicarbonate buffer (pH 9.2) and then combinedwith a solution containing 0.5 mg of AlexaFluor 555 NHS ester in 200 μlformamide. The reaction was incubated at room temperature for 2 hoursand the labelled products were purified on a 20% denaturingpolyacrylamide gel.

High-Throughput Screening

High-throughput screening was carried out using a library of 10,000compounds from the following collections: NCI Oncology (85 compounds),NCI Diversity (1356 compounds), ENZO kinase inhibitors (80 compounds),640 FDA approved drugs, ENZO phosphatase inhibitors (33 compounds),BML-ENZO ion channel ligands (72 compounds), BML-metabotropicglutamatergic ligands (56 compounds), BML nuclear receptor ligands (76compounds), protease inhibitors (53 compounds) and additional compoundsfrom ChemBridge MW and ChemDiv to bring the total number of compounds to10000.

The D135 ribozyme (Su et al., 2005, Nucleic Acids Res, 33:6674-6687),fluorescently labeled oligo substrate 17/2 DL (FIG. 1A-FIG. 1D) (both at20 nM final concentrations in 50 mM MOPS, pH 7.0, 100 mM MgCl₂, 0.5MKCl), and the test compounds (final concentration of 10 μM in 50 mMMOPS, pH 7.0, 100 mM MgCl₂, 0.5M KCl) were aliquoted onto blacknon-binding 384-well plates (Corning 3757) using the multidrop combi(ThermoFisherScientific) and pintool (V & P Scientific). The reactionmixtures were incubated at 37° C. for 45 minutes, then the reaction wasquenched with 100 mM EDTA and fluorescence intensity was analyzed on aTecan Infinite multimode plate reader (λ_(ex) 520 nm, λ_(em) 560 nm, 5nm bandwidth). Data were normalized to untreated wells and wells lackingthe D135 ribozyme, and percent of inhibition was calculated usingActivityBase (IDBS). The Z′-factor was calculated as described (Zhang etal., 1999, J Biomol Screen, 4:67-73):

Z′=1−3×(σ_(p) +σn)/|μ_(p)−μ_(n)|,

-   -   where μ_(p) and σ_(n) are mean value and standard deviation for        the positive control (substrate only), and where μ_(n) and σ_(n)        are mean value and standard deviation for the negative control        (no compound). The average Z′-factor from the screen was        0.83±0.03.

IC₅₀ Measurement for the Small Molecule Inhibition of the D135 RibozymeCleavage Reaction.

Black 96-well plates (Corning 3695) were filled with 50 μl of solutioncontaining 20 nM D135 ribozyme, 20 nM double-labeled substrate 17/2 DLand small molecule inhibitor in 50 mM MOPS, pH 7.0, 100 mM MgCl₂ and 500mM KCl. Small molecule inhibitors were tested at 19 differentconcentrations ranging from 5 nM to 1 mM. Plates were incubated at 37°C. for 10 minutes, then reaction mixtures were quenched with 100 mM EDTAand analyzed on the Synergy H1 plate reader (BioTek). Each experimentwas performed in triplicate. Data were fit to a 4-parameter logisticfunction c+(d−c)/(1+(x/a)^(b)), where a is the IC₅₀, b is the slopeparameter, c is the minimum response and d is the maximum response. Dataare reported as average±s.e.m

Determination of K_(i) Values for Small Molecule Inhibition of theSelf-Splicing Reaction.

Internally labeled SE RNA (2 nM) was incubated with variousconcentrations of inhibitor compound under near-physiological conditions(in 50 mM MOPS, pH 7.5, 8 mM MgCl₂, 100 mM KCl) at 30° C. Aliquots atdifferent time points were quenched and analyzed on 5% denaturingpolyacrylamide gel as previously described (Daniels et al., 1996,Journal of molecular biology, 256:31-49). Data were fit to asingle-exponential equation to determine the first order rate constants(k_(obs)). The latter were then plotted against the concentration of theinhibitor and fit to the equation for non-competitive inhibition todetermine K_(i) values:

k _(obs) =k _(max)/(1+[I]/K _(i)),

where k_(obs) and k_(max) are the first order rate constants measured inthe presence and in the absence of the inhibitor, respectively, [I] isthe concentration of the inhibitor and Ki is the inhibition constant.Experiments were performed four times for API-014, three times forAPY-090, Intronistat A, and Intronistat B and twice for the remainingcompounds to ensure reproducibility. Data represent average±s.e.m.

Effect of Excess of Various RNAs on Splicing Inhibition.

Internally labeled SE RNA (2 nM final concentration) and unlabeled SE,D1, D3, D56 RNAs, yeast tRNA Phe and U2-U6 RNA oligo (2 μM finalconcentrations) were preincubated separately in 50 mM MOPS, pH 7.5, 100mM KCl and 5 mM MgCl₂ (10 mM MgCl2 for the tRNA) at 30° C. for 20minutes. Then the labeled SE RNA and competitor RNA solutions were mixedtogether with simultaneous addition of 19 (Intronistat B) (300 nM finalconcentration) to initiate the reaction. Reaction was carried out undernear-physiological conditions (in 50 mM MOPS, pH 7.5, 8 mM MgCl₂, 100 mMKCl) at 30° C. Aliquots at different time points were quenched andanalyzed on an 5% denaturing polyacrylamide gel as previously described(Daniels et al., 1996, J Mol Biol, 256:31-49). Data were fit with asingle-exponential equation to determine the first order rate constants(k_(obs)). Experiments were performed in triplicate to ensurereproducibility.

Testing Reversibility of Compound Binding.

To examine reversibility of compound binding, the splicing reaction wasinitiated at high concentration (2 μM) of the indicated inhibitor,allowed to react for an hour under near physiological conditions (seeabove), and then diluted by 10-fold with reaction buffer. Aliquots atdifferent time points were quenched and analyzed on an 5% denaturingpolyacrylamide gel as previously described (Daniels et al., 1996, J MolBiol, 256:31-49). Splicing efficiency was observed to significantlyincrease upon dilution of the inhibitor, with a change in rate that iscommensurate with the reduction in compound concentration, indicatingthat binding of the inhibitors to the intron RNA is reversible (FIG.25D). Experiments were performed in duplicate to ensure reproducibility.

Inhibition of Group I Intron Splicing.

Splicing of the Azo-Pre-tRNA intron was carried out essentially aspreviously described (Tanner et al., 1996, RNA 2:74-83). Internallylabeled Azo-Pre-tRNA (20 nM) was incubated in 25 mM HEPES, pH 7.5, 10 mMMgCl₂ at 50° C. for 10 minutes, and then at 32° C. for 2 minutes. ThenIntronistat B was added to the final concentration of 1, 5, 10, 20, 50and 10011M and reaction was initiated by addition of 100 μM GTP (finalconcentration). The final concentration of DMSO in all samples was 10%.Aliquots at different time points were quenched and analyzed on an 5%denaturing polyacrylamide gel as previously described (Tanner et al.,1996, RNA 2:74-83; Daniels et al., 1996, J Mol Biol, 256:31-49)). Allexperiments were performed in triplicate to ensure reproducibility. Datarepresent average±s.e.m.

Yeast Respiration Assay in S. cerevisiae.

Yeast respiration assays were conducted either in liquid YPD media (BDBacto Yeast Extract, BD Bacto Peptone, 2% glucose) or in YPGE media (BDBacto Yeast Extract, BD Bacto Peptone, 3% glycerol, 3% ethanol),essentially as described for the antifungal MIC assays (see below). Toinitiate these experiments, 100 μl of a fresh stock solution wasprepared for each compound (3.2 mg/ml for Amphotericin B and 12.8 mg/mlfor each test compounds) in DMSO. This concentrated stock solution wasplaced in the first well of 12-well row on a 96-well plate. Thesubsequent nine wells in this row contained 50 μl of DMSO for serialdilution. From the first well, 50 μl of the compound stock waswithdrawn, transferred to the second well and mixed. This process wasrepeated from the second and subsequent wells, resulting in a 1:2 serialdilution of the stock into DMSO. Wells 11 and 12 contained DMSO only, tobe used as no compound control and sterility control, with no inoculumadded, respectively. After preparing this plate, 2.5 μl from each of the12 wells was transferred to the wells of a separate 96-well plate, eachof which contained 122.5 μl of YPD or YPGE medium, resulting in a set ofserial dilutions of compound into growth media in a volume of 125 μl.Then 50 μl from each of these dilutions were transferred onto a third96-well plate (the assay plate) and mixed with an equal volume (50 μl)of freshly prepared yeast inoculum in YPD or YPGE medium set to provide0.5×10³ to 2.5×10³ colony forming units per 1 mL (wells 1-11), orYPD/YPGE alone (sterility control, well 12). The final compoundconcentration range of the assay was 0.25-128 μg/ml for test compoundsand 0.0625-32 μg/ml for Amphotericin B. The final concentration of DMSOwas 1%. Plates were incubated at 30° C. and visually analyzed forturbidity after 24 and 48 hours of incubation. The lowest compoundconcentration at which no growth was apparent is reported as the MIC(Table 1). All experiments were performed in triplicate.

TABLE 1 MIC of candidate compounds. MIC in YPD MIC in YPGE Compound(glucose), μg/ml (glycerol), μg/ml APY-019 >128 64 APY-014 >128 >128APY-081 >128 32 APY-084 >128 16-32 APY-090 >128 64 APY-093 >128 >128APY-101 (Intronistat A) >128 32 NED-2020 (Intronistat B) >128 64Amphotericin B 2 16S. cerevisiae Growth Assays

Cultures of S. cerevisiae (Wild-type: NP40-36a, mtDNA intronless: XPM46(Perez-Martinez et al., 2003, EMBO J, 22:5951-5961) were grown overnightwith shaking to mid-log phase in YPGE at 30° C. Equal numbers of cellswere harvested and serially-diluted 1:5. Cells were then plated on YPDor YPGE media containing DMSO or Intronistat A dissolved in DMSO (finalconcentration: 64 or 128 μg/mL). Plates were grown for one day (YPDplates), two days (YPGE DMSO plate), or 18 days (YPGE 18 (Intronistat A)plate) at 30° C. Experiment was replicated twice to ensurereproducibility of growth phenotype.

Analysis of Group II Intron Splicing in S. cerevisiae by RT-qPCR.

A culture of S. cerevisiae was grown overnight with shaking in YPGE at30° C. The saturated culture was then diluted and grown to mid-log phasein YPGE. Equal volumes of DMSO or compounds dissolved in DMSO were addedto individual cultures (final concentrations of NED-2020: 64 μg/mL,APY-101: 32 μg/mL, APY-081: 32 μg/mL, APY-014: 64 μg/mL, Amphotericin B:8 μg/mL), and incubated for four hours. Cells were harvested bycentrifugation, washed with 500 μL ice-cold MilliQ water, centrifugedagain, and snap-frozen in liquid nitrogen. Total RNA was isolated usingE.Z.N.A. Yeast Purification Kit (Omega Bio-tek) according tomanufacturer's procedures. The RNA was eluted in 50 μL ofRNase/DNase-free water (ThermoFisher), followed by DNAse treatment withRQ1 DNase (Promega), for 1 hour at 37° C. Then it was mixed with 6 μl of3M NaOAc and precipitated with 75% EtOH at −20° C. overnight. Then theRNA was reverse-transcribed with SuperScript III (ThermoFisher)following manufacturer's recommendations with 200 ng Random HexamerPrimers (ThermoFisher) and RNasin (ThermoFisher) in a 20 μl reaction.After reverse transcription, RNA was degraded by addition of 2N NaOH (2μl) and incubation at 95° C. for 5 minutes. After cooling on ice for 5minutes, 2 μl of 1M HCl and 2 μl of 3M NaOAc were added to the reactionmixture, and cDNA was precipitated with 75% EtOH at −20° C. for 30minutes and resuspended in RNase/DNase-free water. The cDNA levels werequantified with real-time PCR using LightCycler 480 SYBR Green I (Roche)and a CFX384 Real-Time PCR Detection System (Bio-Rad) in triplicate.Relative levels of indicated RNA species from different conditions werenormalized according to the ΔΔC_(T) method via the following equation(Livak and Schmittgen, 2001, Methods, 25:402-408):

ΔΔC _(T)=[(C _(T) COX1total or unspliced−C _(T) ACT1or PGK1)testcompound−(C _(T) COX1total or unspliced−C _(T) ACT1or PGK1)DMSO)].

The average and SEM of 2{circumflex over ( )}-ΔΔC_(T) is reported foreach sample from three independent replicates. The following primer setswere used to amplify indicated targets: ACT1: TCGAACAAGAAATGCAAACCG (SEQID NO:1), GGCAGATTCCAAACCCAAAAC (SEQ ID NO:2); PGK1:TGTCTTGGCTTCTCACTTGG (SEQ ID NO:3), TTCAACTTCTGGACCGACAC (SEQ ID NO:4);Total COX1: TGGTATGCCTAGAAGAATTCCTG (SEQ ID NO:5),AGAATAATGATAATAGTGCAATGAATGAAC (SEQ ID NO:6); Unspliced COX1:CTTACTACGTGGTGGGACATT (SEQ ID NO:7), GTCATTACAGCTTAGCATATTTATGT (SEQ IDNO:8). In the course of analysis, no reverse transcription controlsamples displayed minimal signal, indicating that amplicons wereamplified from cDNA and not genomic DNA, and gel electrophoresisconfirmed amplification of the intended amplicon.

Analysis of Group II Intron Splicing in C. parapsilosis by qRT-PCR.

A culture of C. parapsilosis was grown overnight with shaking in RPMI at30° C. The saturated culture was then diluted and grown to mid-log phasein RPMI. Equal volumes of DMSO or compounds dissolved in DMSO were addedto individual cultures (final concentrations of Intronistat B and 4: 32μg/mL) and incubated for two hours. Potassium cyanide (KCN), aninhibitor of complex IV of the electron transport chain, was added to afinal concentration of 10 mM for an additional two hours to inducenascent COX1 expression. Cells were harvested, RNA prepared andreverse-transcribed, and cDNA quantified as in S. cerevisiaeexperiments. The mean and s.e.m. of 2{circumflex over ( )}-DDCT isreported for each sample from three independent replicates. Thefollowing primer sets were used to amplify indicated targets:

C.p.PGK1: (SEQ ID NO: 13) TGGATGGGTCTTGATTGTGG, (SEQ ID NO: 14)GTCAAATTCAAAGACACCCGG; total C.p.COX1: (SEQ ID NO: 15)GGTGCTGTAGATATGGCATTTG, (SEQ ID NO: 16) GCACTAATTGATGATAGTGGAGGA;unspliced C.p.COX1: (SEQ ID NO: 17) TGTTCTTGTTACTGGTCATGCT,(SEQ ID NO:18) AGCACTTACTAACTGTTCACGTC.Determination of MICs for the Small Molecule Inhibitors Against C.parapsilosis

Minimal inhibitory concentrations (MICs) for C. parapsilosis weredetermined in a final volume of 100 μl. Fresh stock solutions of controlantifungal drugs (Amphotericin B and Itraconazole) and of test compoundswere prepared at 3.2 mg/ml and at 12.8 mg/ml respectively in DMSO. Afterplacing the stock in the first well, solutions of test compounds andantifungal controls were serially diluted 1:2 with DMSO into the firstnine successive wells of a 12-well row on a 96-well plate (as describedabove). After dilution, 2.5 μl from each of these 10 wells weretransferred onto a separate 96-well plate and diluted with 122.5 μl ofRPMI-1640 medium (Sigma). Wells 11 and 12 contained 2.5 μl DMSO and122.5 μl of RPMI-1640 as a positive growth control (no compound) andsterility control (no inoculum added). A 50 μl volume was taken fromeach of the RPMI dilutions (wells 1-12) and transferred to the wells ofa third 96-well plate (assay plate), where they were mixed with an equalvolume (50 μl) of freshly prepared inoculum of C. parapsilosis inRPMI-1640 medium, set at a density to provide the 0.5×10³ to 2.5×10³colony forming units per 1 mL (except for the sterility control).Sterility control was mixed with 50 μl of sterile medium.

The final concentration range of the assay was 0.25-128 μg/ml for testcompounds and 0.0625-32 μg/ml for Amphotericin B and Itraconazole. Thefinal concentration of DMSO was 1%. The assay plate was incubated at 35°C. or 37° C. and visually analyzed for turbidity after 24 and 48 hoursof incubation. The lowest compound concentration at which no growth wasapparent is reported as the MIC. All experiments were performed intriplicate.

Cytotoxicity in HEK-293T Cells.

HEK-293T cells were cultured in Dulbecco's Modified Eagle's Medium(DMEM) (Gibco) supplemented with 10% Fetal Bovine Serum (FBS, Gibco) and100 U/ml of Penicillin-Streptomycin (Gibco) at 37° C. and 5% CO₂. Forthe cytotoxicity experiments, cells were aliquoted into black 96-wellplates with a clear bottom (Corning 3603) at a concentration of 10,000cells per well. The cells were grown at 37° C., 5% CO₂ for 5-6 hours,then the medium was replaced with the same medium without FBS and cellswere grown at 37° C., 5% CO₂ for 24 hours. Freshly prepared stocksolutions of test compounds (12.8 mg/ml in DMSO) were serially diluted1:2 with DMSO into the first 11 successive wells of a 12-well row on a96-well plate as described above. Well 12 was used as a growth control(no compound). After dilution, 1 μl from each well of the compound platewas added to the plate containing cells (the assay plate), and cellswere incubated at 37° C., 5% CO₂ for another 24 hours. After incubation,cell viability was determined using the luminescent Cell Titer Glo cellviability assay (Promega), in which 100 μl of the assay reagent wasadded to each well of the assay plate. After gentle shaking for 7-10minutes, the plates were analyzed on a Synergy H1 plate reader (BioTek).Luminescence was plotted against the compound concentration and IC₅₀values were determined by fitting the data to a 4-parameter logisticfunction c+(d−c)/(1+(x/a)^(b)), where a is the IC₅₀, b is the slopeparameter, c is the minimum response and d is the maximum response.Experiments were replicated to ensure reproducibility.

Statistics and Reproducibility

The following reported data represent average of n=3 independentexperiments: all IC₅₀ values for ribozyme cleavage, Ki values forcompounds APY-090, Intronistat A and Intronstat B, k_(obs) values forinhibition of splicing in the presence of the excess of various RNAs,time courses for inhibition of the group I intron splicing, DDCT valuesobtained from qRT-PCR analysis of group II intron splicing in C.parapsilosis, MIC values from yeast respiration assay, MIC values forsmall molecules against C. parapsilosis, IC₅₀ values for cytotoxicity inHEK-293T cells for compounds CAS 3260-50-2, APY-019, APY-083, APY-077,APY-084, APY-090, APY-094 and APY-097. The following reported datarepresent average of n=4 independent experiments: Ki for APY-014, DDCTvalues obtained from qRT-PCR analysis of group II intron splicing in S.cerevisiae, IC₅₀ values for cytotoxicity in HEK-293T cells for compoundsAPY-007, APY-014, APY-098, APY-024, APY-068, APY-093, APY-081, APY-091,Intronistat A and Intronstat B. The following experiments were repeatedtwice to ensure reproducibility: test of reversibility of compoundbinding, S. cerevisiae growth assays, cytotoxicity of APY-001 inHEK-293T cells, Ki determination for compounds CAS 3260-50-2, APY-007,APY-001, APY-098, APY-019, APY-024, APY-083, APY-068, APY-077, APY-093,APY-081, APY-084, APY-091, APY-094 and APY-097. All values are reportedas mean±s.e.m.

Synthesis of Small Molecules

Unless otherwise stated, all reagents were purchased from commercialsuppliers and used without further purification. Analytical thin layerchromatography (TLC) was performed on Merck Millipore precoated (0.25 mmthickness) silica gel plates containing F254 indicator. Visualizationwas accomplished by irradiation with UV light at 254 nm or PMA or KMnO₄stain solution. Flash column chromatography was performed on SiliaFlash®F60 silica gel (40-63 μm) supplied by Silicycle. All ¹H NMR spectra wererecorded on an Agilent DD2 400 MHz spectrometer (400 MHz) in deuteratedsolvent and ¹³C NMR spectra were recorded on an Agilent DD2 600 MHzspectrometer (151 MHz) in deuterated solvent. LCMS were run using anAdvion Expression® mass spectrometer with an Agilent Technologies 1260Infinity® liquid chromatograph front end.

General Procedure for the Removal of Benzyl Groups from3,4,5-Tribenzyloxybenzenes (Procedure A)

A cooled (−70° C.) stirred solution of tribenzyloxybenzene (0.25 mmol)in anhydrous DCM (5 mL) under nitrogen was treated dropwise with 1NBBr3/DCM (1.5 mL, 1.5 mmol), allowed to warm to room temperature andstirred at room temperature for 4 hours, then recooled (−60° C.). Themixture was quenched dropwise with methanol (5 mL), warmed to roomtemperature, stirred for 30 minutes, then concentrated in vacuo.

Preparation of 2-Benzoylbenzofurans5-Bromobenzofuran-3-yl)(3,4,5-tris(benzyloxy)phenyl)methanone(Intermediate 1)

A stirred suspension of potassium carbonate (1.04 g, 7.5 mmol) in2-butanone (10 mL) under nitrogen was treated with a solution of5-bromosalicylaldehyde (1.01 g, 5 mmol) in 2-butanone (10 mL) andstirred for 15 minutes. A solution of2-bromo-1-(3,4,5-tris(benzyloxy)phenyl)ethan-1-one (2.59 g, 5 mmol) in2-butanone (30 mL) was added, and the mixture heated to 50° C. for 20hours, cooled to room temperature, and diluted with water (150 mL). Theaqueous suspension was stirred a few minutes, filtered, and the filtercake rinsed with water and dried in vacuo to afford 3.02 g (97%) of(5-bromobenzofuran-3-yl)(3,4,5-tris(benzyloxy)phenyl)methanone as a paletan voluminous solid (FIG. 2 ). ¹H NMR (CDCl₃): δ 7.76 (d, J=2 Hz, 1H),7.55 (dd, J=9 Hz, 2 Hz, 1H), 7.45 (m, 3H), 7.35-7.43 (m, 10H), 7.27-7.32(m, 5H), 5.21 (s, 2H), 5.19 (s, 4H). ¹³C NMR (CDCl₃): δ 182.61, 154.44,152.64, 152.46, 142.82, 137.29, 136.62, 131.62, 131.14, 128.77, 128.68,128.56, 128.26, 128.08, 128.06, 127.33, 125.75, 116.88, 115,04, 114.01,109.38, 75.23, 71.15. LCMS m/z: [M+H]⁺620.2 (20%).

Benzofuran-2-yl(3,4,5-trihydroxyphenyl)methanone (APY-068)

A stirred suspension of potassium carbonate (100 mg, 0.724 mmol) in2-butanone (1 mL) under nitrogen was treated with a solution ofsalicylaldehyde (61 mg, 0.50 mmol) in 2-butanone (1 mL) and stirred 15minutes. A solution of2-bromo-1-(3,4,5-tris(benzyloxy)phenyl)ethan-1-one (259 mg, 0.5 mmol) in2-butanone (3 mL) was added, and the mixture heated to 50° C. for 20hours, cooled to room temperature, and diluted with water (15 mL). Themixture was extracted with ethyl acetate (40 mL, then 2×15 mL) and thecombined organic solution was washed with water and brine (25 mL each),dried (MgSO₄) and concentrated in vacuo. The residual solid wasdissolved in heptane/dichloromethane and loaded onto a silica gel columnand eluted with 60% DCM/heptane, then 80% DCM/heptane, then DCM toafford 223 mg (83%) of tribenzyloxy penultimate intermediate as a whitesolid. This compound (200 mg, 0.37 mmol) was taken directly throughProcedure A to afford the crude product, which was dissolved indichloromethane containing a little ethyl acetate, loaded onto a silicagel column (˜60 cc), and eluted with 2:1 DCM/ethyl acetate to afford 44mg (44%) of benzofuran-2-yl(3,4,5-trihydroxyphenyl)methanone as a paleorange solid (FIG. 3 ). ¹H NMR (d6-acetone): δ 8.38 (br s, 2H), 8.27 (brs, 1H), 7.84 (d, J=8 Hz, 1H), 7.67 (d, J=8 Hz, 1H), 7.63 (s, 1H), 7.53(t, J=7.5 Hz, 1H), 7.37 (t, J=7.5 Hz, 1H), 7.28 (s, 2H). ¹³C NMR(d4-acetic acid): δ 181.78, 155.52, 152.79, 145.33, 138.50, 128.16,127.80, 127.20, 123.83, 123.29, 114.77, 111.99, 109.38. LCMS m/z: [M−H]⁻269.7 (100%).

5-[(5,7-Dichloro-1-benzofuran-2-yl)carbonyl]benzene-1,2,3-triol(APY-083)

A stirred suspension of potassium carbonate (100 mg, 0.724 mmol) in2-butanone (1 mL) under nitrogen was treated with a solution of3,5-dichlorosalicylaldehyde (95.5 mg, 0.50 mmol) in 2-butanone (1 mL)and stirred 15 minutes. A solution of2-bromo-1-(3,4,5-tris(benzyloxy)phenyl)ethan-1-one (259 mg, 0.5 mmol) in2-butanone (3 mL) was added, and the mixture heated to 50° C. for 20hours, cooled to room temperature, and diluted with water (15 mL). Theaqueous suspension was stirred a few minutes, filtered, and the filtercake rinsed with water and dried to afford 284 mg (92%) of a pale tansolid which was taken forward without purification. A cooled (−70° C.)stirred solution of this penultimate intermediate (284 mg, 0.466 mmol)in anhydrous DCM (8 mL) under nitrogen was subjected to Procedure A with1N BBr3/DCM (2.8 mL, 2.8 mmol), and the resultant dark solid wasdissolved in 10% methanol/DCM, loaded onto a small silica column (˜40cc) and eluted with 10% methanol/DCM to afford a brown solid, which wastriturated from cold DCM to afford 95 mg (60%) of5-[(5,7-dichloro-1-benzofuran-2-yl)carbonyl]benzene-1,2,3-triol as agray solid (FIG. 4 ). ¹H NMR (d6-acetone): δ 8.45 (br s, 2H), 8.39 (brs, 1H), 7.88 (d, J=2 Hz, 1H), 7.68 (s, 1H), 7.65 (d, J=2 Hz, 1H), 7.28(s, 2H). ¹³C NMR (d6-acetone): δ 181.13, 154.48, 149.86, 145.44, 139.01,129.86, 129.07, 127.58, 127.10, 121.69, 117.77, 114.38, 109.48. LCMSm/z: [M−H]⁻ 336.5 (100%), 338.5 (70%).

5-{[5-(4-Methylpyridin-3-yl)-1-benzofuran-2-yl]carbonyl}benzene-1,2,3-triol(APY-081)

A stirred mixture of(5-bromobenzofuran-3-yl)(3,4,5-tris(benzyloxy)phenyl)methanone (186 mg,0.30 mmol), 4-methylpyridine-3-boronic acid (58 mg, 0.42 mmol) andpotassium carbonate (166 mg, 1.2 mmol) in 9:1 dioxane/water (3 mL) in apressure tube was degassed over 20 minutes with argon, then treated with1,1-bis(diphenylphosphino)ferrocene dichloropalladium (11) (25 mg, 0.034mmol), capped, and heated to 90° C. for 18 hours and cooled to roomtemperature and diluted with ethyl acetate (12 mL). The mixture wascombined with water (10 mL) and separated. The aqueous solution wasextracted with ethyl acetate (8 mL) and the combined organic solutionwas washed with brine (15 mL), dried (MgSO4) and concentrated in vacuo.The dark residue was dissolved in DCM and loaded onto a silica gelcolumn (˜75 cc) and eluted with 20%, then 25% EtOAc/DCM to afford 160 mg(84%) of the penultimate intermediate as a pale yellow viscous oil. Acooled (−70° C.) stirred solution of this compound (160 mg, 0.253 mmol)in anhydrous DCM (5 mL) under nitrogen was subjected to Procedure A with1N BBr3/DCM (1.5 mL, 1.5 mmol), and the resultant tan solid wastriturated from acetonitrile, collected, and dried in vacuo to afford 91mg (99%) of5-{[5-(4-methylpyridin-3-yl)-1-benzofuran-2-yl]carbonyl}benzene-1,2,3-triolas a pale tan solid (FIG. 5 ). ¹H NMR (d6-DMSO): δ 9.40 (br s, 3H), 8.83(s, 1H), 8.79 (d, J=6 Hz, 1H), 8.01 (d, J=6 Hz, 1H), 7.97 (m, 1H), 7.92(d, J=8.5 Hz, 1H), 7.73 (br s, 1H), 7.65 (dd, J=8.5 Hz, 2 Hz, 1H), 7.09(s, 2H), 2.50 (s, 3H). ¹³C NMR (d6-DMSO): δ 182.07, 156.49, 155.28,153.35, 146.19, 142.34, 140.91, 139.98, 139.86, 130.72, 129.68, 128.49,127.64, 126.95, 124.95, 115.38, 113.01, 109.57, 21.23. LCMS m/z: [M−H]⁻360.0 (100%). LCMS m/z: [M+H]⁺ 362.0 (100%).

5-({5-[4-(Hydroxymethyl)phenyl]-1-benzofuran-2-yl}carbonyl)benzene-1,2,3-triol(APY-084)

A stirred mixture of(5-bromobenzofuran-3-yl)(3,4,5-tris(benzyloxy)phenyl)methanone (248 mg,0.40 mmol), 4-hydroxymethylphenylboronic acid (85 mg, 0.56 mmol) andpotassium carbonate (221 mg, 1.6 mmol) in 9:1 dioxane/water (4 mL) in apressure tube was degassed over 20 minutes with argon, then treated with1,1-bis(diphenylphosphino)ferrocene dichloropalladium (II) (33 mg, 0.045mmol), capped, and heated to 90° C. for 18 hours and cooled to roomtemperature and diluted with ethyl acetate (12 mL). The mixture wascombined with water (10 mL) and separated. The aqueous solution wasextracted with ethyl acetate (8 mL) and the combined organic solutionwas washed with brine (15 mL), dried (MgSO4) and concentrated in vacuo.The dark residue was dissolved in DCM and loaded onto a silica gelcolumn (˜75 cc) and eluted with 3%, then 5% EtOAc/dichloromethane toafford 183 mg (71%) of the penultimate intermediate as a white solid. Acooled (−70° C.) stirred solution of this compound (174 mg, 0.269 mmol)in anhydrous DCM (5 mL) under nitrogen was subjected to Procedure A with1N BBr3/DCM (2.15 mL, 2.15 mmol), and the resultant solid dissolved in9:1 DCM/methanol and added to a silica gel column (˜70 cc). The columnwas eluted with 9:1 DCM/methanol to afford a brown solid, which wastriturated from DCM and dried to afford 70 mg (69%) of5-({5-[4-(hydroxymethyl)phenyl]-1-benzofuran-2-yl}carbonyl)benzene-1,2,3-triolas a yellow-tan solid (FIG. 6 ). ¹H NMR (d6-acetone): δ 8.38 (br s, 3H),8.11 (d, J=1.5 Hz, 1H), 7.85 (dd, J=9 Hz, 1.5 Hz, 1H), 7.66-7.79 (m,4H), 7.59 (d, J=8 Hz, 2H), 7.30 (s, 2H), 4.72 (s, 2H), 3.14 (br s, 1H).¹³C NMR (d6-acetone): δ 182.30, 155.25, 153.38, 145.39, 140.70, 137.37,136.61, 129.65, 127.80, 127.58, 127.36, 127.30, 121.28, 115.17, 112.23,109.18, 99.32, 32.98. LCMS m/z: [M−H]⁻ 374.9 (50%).

5-[(5-{4-[(Diethylamino)methyl]phenyl}-1-benzofuran-2-yl)carbonyl]benzene-1,2,3-triolhydrobromide (APY-090)

A stirred mixture of(5-bromobenzofuran-3-yl)(3,4,5-tris(benzyloxy)phenyl)methanone (186 mg,0.30 mmol),diethyl({[4-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methyl})amine(122 mg, 0.42 mmol) and potassium carbonate (166 mg, 1.2 mmol) in 9:1dioxane/water (3 mL) in a pressure tube was degassed over 20 minuteswith argon, then treated with 1,1-bis(diphenylphosphino)ferrocenedichloropalladium (II) (25 mg, 0.034 mmol), capped, and heated to 90° C.for 18 hours and cooled to room temperature and diluted with DCM (12 mL)and filtered through Celite®. The residual dark oil was dissolved inethyl acetate and loaded onto a silica gel column (˜50 cc) and elutedwith EtOAc, then 5% methanol/EtOAc to afford 187 mg (89%) of thepenultimate intermediate as an amber solid. A cooled (−70° C.) stirredsolution of this compound (187 mg, 0.266 mmol) in anhydrous DCM (5 mL)under nitrogen was subjected to Procedure A with 1N BBr3/DCM (1.6 mL,1.6 mmol), and the resultant solid triturated from DCM to afford 100 mg(73%) of5-[(5-{4-[(diethylamino)methyl]phenyl}-1-benzofuran-2-yl)carbonyl]benzene-1,2,3-triolhydrobromide as a tan solid (FIG. 7 ). ¹H NMR (d6-DMSO): δ 9.20-9.50 (m,3H), 7.80-8.20 (m, 4H), 7.62-7.70 (m, 2H), 7.38-7.55 (m, 2H), 7.09 (brs, 2H), 4.36 (d, J=6 Hz, 1H), 4.20-4.30 (m, 1H), 2.97-3.17 (m, 4H),1.15-1.30 (m, 6H). ¹³C NMR (d6-DMSO): δ 182.11, 155.20, 153.19, 146.18,141.42, 139.88, 135.97, 132.33, 132.16, 129.60, 127.99, 127.83, 127.06,121.93, 115.70, 113.11, 109.53, 54.95, 46.35, 8.84. LCMS m/z: [M−H]⁻429.7 (100%). LCMS m/z: [M+H]⁺ 431.7 (100%).

N,N-Diethyl-4-{2-[(3,4,5-trihydroxyphenyl)carbonyl]-1-benzofuran-5-yl}benzamide(APY-091)

A stirred mixture of(5-bromobenzofuran-3-yl)(3,4,5-tris(benzyloxy)phenyl)methanone (186 mg,0.30 mmol), [4-(diethylcarbamoyl)phenyl]boronic acid (93 mg, 0.42 mmol)and potassium carbonate (166 mg, 1.2 mmol) in 9:1 dioxane/water (3 mL)in a pressure tube was degassed over 20 minutes with argon, then treatedwith 1,1-bis(diphenylphosphino)ferrocene dichloropalladium (II) (25 mg,0.034 mmol), capped, and heated to 90° C. for 18 hours and cooled toroom temperature and diluted with DCM (12 mL) and filtered throughCelite®. The residual dark oil was dissolved in DCM and loaded onto asilica gel column (˜75 cc) and eluted with 5% EtOAc/DCM, then 10%EtOAc/DCM to afford 167 mg (78%) of the penultimate intermediate as apale amber solid. A cooled (−70° C.) stirred solution of this compound(167 mg, 0.233 mmol) in anhydrous DCM (5 mL) under nitrogen wassubjected to Procedure A with 1N BBr3/DCM (1.85 mL, 1.85 mmol), and theresultant dark solid was dissolved in 9:1 DCM/methanol and loaded ontosilica gel (˜40 cc) and eluted with DCM, then 9:1 DCM/methanol to afford69 mg (66%) ofN,N-diethyl-4-{2-[(3,4,5-trihydroxyphenyl)carbonyl]-1-benzofuran-5-yl}benzamideas a brown glassy solid (FIG. 8 ). ¹H NMR (d6-DMSO): δ 9.36 (br s, 3H),8.12 (s, 1H), 7.83 (br s, 2H), 7.74 (d, J=7.5 Hz, 2H), 7.69 (br s, 1H),7.43 (d, J=7.5 Hz, 2H), 7.10 (br s, 2H), 3.10-3.50 (m, 4H), 1.09 (m,6H). ¹³C NMR (d6-DMSO): δ 182.14, 170.12, 155.16, 153.14, 146.20,140.94, 139.90, 136.59, 136.16, 127.99, 127.75, 127.38, 127.33, 127.09,121.89, 115.77, 113.06, 109.53, 49.04, 43.30, 14.54, 13.28. LCMS m/z:[M−H]⁻ 443.5 (100%). LCMS m/z: [M+H]+446.6 (100%).

N-Butyl-4-{2[(3,4,5-trihydroxyphenyl)carbonyl]-1-benzofuran-5-yl}benzene-1-sulfonamide(APY-094)

A stirred mixture of(5-bromobenzofuran-3-yl)(3,4,5-tris(benzyloxy)phenyl)methanone (186 mg,0.30 mmol), [4-(butylsulfamoyl)phenyl]boronic acid (108 mg, 0.42 mmol)and potassium carbonate (166 mg, 1.2 mmol) in 9:1 dioxane/water (3 mL)in a pressure tube was degassed over minutes with argon, then treatedwith 1,1-bis(diphenylphosphino)ferrocene dichloropalladium (II) (25 mg,0.034 mmol), capped, and heated to 90° C. for 18 hours and cooled toroom temperature and diluted with DCM (12 mL) and filtered throughCelite®. The residual dark oil was dissolved in DCM and loaded onto asilica gel column (˜80 cc) and eluted with 2% EtOAc/DCM, then 3%EtOAc/DCM to afford 189 mg (84%) of the penultimate intermediate as awhite solid. A cooled (−70° C.) stirred solution of this compound (185mg, 0.246 mmol) in anhydrous DCM (5 mL) under nitrogen was subjected toProcedure A with 1N BBr3/DCM (2.0 mL, 2.0 mmol), and the resultantmaterial was dissolved in 10% methanol/DCM and added to a silica gelcolumn (˜90 cc) and eluted with 10% methanol/DCM to afford 89 mg (75%)ofN-butyl-4-{2[(3,4,5-trihydroxyphenyl)carbonyl]-1-benzofuran-5-yl}benzene-1-sulfonamideas a brown solid (FIG. 9 ). ¹H NMR (d6-acetone): δ 8.38 (m, 3H), 8.18(br s, 1H), 7.84-8.00 (m, 5H), 7.80 (d, J=9 Hz, 1H), 7.70 (br s, 1H),7.30 (s, 2H), 6.46 (t, J=6 Hz, 1H), 2.95 (q, J=7 Hz, 2H), 1.48 (m, 2H),1.33 (m, 2H), 0.85 (t, J=7 Hz, 3H). ¹³C NMR (d6-acetone): δ 181.66,155.54, 153.66, 145.38, 144.47, 139.83, 138.64, 135.61, 128.05, 127.99,127.71, 127.52, 127.42, 121.94, 114.87, 112.62, 109.44, 42.76, 31.53,19.49, 12.97. LCMS m/z: [M−H]⁻ 479.5 (80%), 480.5 (100%).

1-(4-{2-[(3,4,5-Trihydroxyphenyl)carbonyl]-1-benzofuran-5-yl}phenyl)ethan-1-one(APY-101)

A stirred mixture of(5-bromobenzofuran-3-yl)(3,4,5-tris(benzyloxy)phenyl)methanone (248 mg,0.40 mmol), 4-acetylphenylboronic acid (92 mg, 0.56 mmol) and potassiumcarbonate (221 mg, 1.6 mmol) in 9:1 degassed dioxane/water (4 mL) in apressure tube was treated with 1,1-bis(diphenylphosphino)-ferrocenedichloropalladium (II) (34 mg, 0.046 mmol), capped under nitrogen, andheated to 90° C. for 18 hours. The solution was cooled to roomtemperature, diluted with DCM (15 mL) and filtered through Celite®. Thefiltrate was concentrated in vacuo and the dark residue was dissolved inDCM and loaded onto a silica gel column (˜100 cc) and eluted with DCM,then 3% EtOAc/DCM to afford 170 mg (65%) of the penultimate intermediateas a white solid. A cooled (−70° C.) stirred solution of this compound(165 mg, 0.251 mmol) in anhydrous DCM (5 mL) under nitrogen wassubjected to Procedure A with 1N BBr3/DCM (2.0 mL, 2.0 mmol), and theresultant solid was dissolved in EtOAc/MeOH. The solution was added to asilica gel column (˜50 cc) and eluted with 15% MeOH/EtOAc to afford abrown solid which was triturated from DCM to afford 80 mg (82%) of1-(4-{2-[(3,4,5-trihydroxyphenyl)carbonyl]-1-benzofuran-5-yl}phenyl)ethan-1-oneas a tan solid (FIG. 10 ). ¹H NMR (d6-acetone): δ 8.50-8.85 (m, 3H),8.18 (m, 1H), 8.10 (d, J=8 Hz, 2H), 7.90 (m, 1H), 7.86 (d, J=8 Hz, 2H),7.80 (d, J=9 Hz, 1H), 7.69 (s, 1H), 7.27 (s, 2H), 2.62 (s, 3H). ¹³C NMR(d6-acetone): δ 196.66, 181.79, 155.49, 153.68, 145.76, 145.05, 138.96,136.00, 135.96, 128.91, 128.01, 127.83, 127.33, 127.29, 121.82, 114.82,112.58, 109.40, 25.92. LCMS m/z: [M−H]⁻ 387.2 (100%). LCMS m/z: [M+H]⁺389.1 (100%).

5-({5-[6-(Trifluoromethyl)pyridin-3-yl]-1-benzofuran-2-yl}carbonyl)benzene-1,2,3-triol(APY-097)

A stirred mixture of(5-bromobenzofuran-3-yl)(3,4,5-tris(benzyloxy)phenyl)methanone (248 mg,0.40 mmol), [6-(trifluoromethyl)pyridin-3-yl]boronic acid (107 mg, 0.56mmol) and potassium carbonate (221 mg, 1.6 mmol) in 9:1 degasseddioxane/water (4 mL) in a pressure tube was treated with1,1-bis(diphenylphosphino)ferrocene dichloropalladium (II) (34 mg, 0.046mmol), capped under nitrogen, and heated to 90° C. for 18 hours andcooled to room temperature and diluted with DCM (15 mL) and filteredthrough Celite®. The dark residue was dissolved in DCM and loaded onto asilica gel column (˜100 cc) and eluted with 1.5% EtOAc/DCM, then 3%EtOAc/DCM to afford 222 mg (81%) of the penultimate intermediate as awhite solid. A cooled (−70° C.) stirred solution of this compound (220mg, 0.321 mmol) in anhydrous DCM (6 mL) under nitrogen was subjected toProcedure A with 1N BBr3/DCM (2.6 mL, 2.6 mmol), and the resultant brownsolid was dissolved in 9:1 DCM/methanol and loaded onto a silica gelcolumn (˜75 cc) and eluted with 10% methanol/EtOAc. The resultant glassysolid was triturated from DCM to afford 101 mg (76%) of5-({5-[6-(trifluoromethyl)pyridin-3-yl]-1-benzofuran-2-yl}carbonyl)benzene-1,2,3-triolas a tan solid (FIG. 11 ). ¹H NMR (d6-acetone): δ 9.10 (s, 1H), 8.39 (m,4H), 8.26 (s, 1H), 7.97 (d, J=8.5 Hz, 2H), 7.85 (m, 1H), 7.72 (s, 1H),7.30 (s, 2H). ¹³C NMR (d6-DMSO): δ 181.63, 155.77, 153.79, 148.60,145.39, 139.36, 138.69, 136.10, 132.47, 128.15, 127.99, 127.43, 122.95,122.38, 121.14, 120.60, 114.72, 112.95, 109.45. LCMS m/z: [M−H]⁻ 414.5(100%). LCMS m/z: [M+H]⁺ 416.3 (100%).

N-(2-(dimethylamino)ethyl)-2-(3,4,5-trihydroxybenzoyl)-benzofuran-5-carboxamide(NED-2018)

An ice cooled stirred solution of2-{[3,4,5-tris(benzyloxy)phenyl]carbonyl}-3a,7a-dihydro-1-benzofuran-5-carboxylicacid (250 mg, 0.43 mmol), 2-dimethylaminoethylamine (45 mg, 0.51 mmol)and HOBT hydrate (81.6 mg, 0.53 mmol) in anhydrous DCM (4 mL) undernitrogen was treated with trimethylamine (0.18 mL, 1.28 mmol) and EDCI(102 mg, 0.53 mmol). Allowed reaction to warm to room temperature andstir overnight (18 h). The resulting reaction was concentrated to anoily residue, which was purified by flash chromatography (10%diethylamine in Ethyl Acetate) to affordN-[2-(dimethylamino)ethyl]-2-{[3,4,5-tris(benzyloxy)phenyl]carbonyl}-3a,7a-dihydro-1-benzofuran-5-carboxamide.

A cooled (−70° C.) stirred solution ofN-[2-(dimethylamino)ethyl]-2-{[3,4,5-tris(benzyloxy)phenyl]carbonyl}-3a,7a-dihydro-1-benzofuran-5-carboxamide(164 mg, 0.25 mmol) in anhydrous dichloromethane (5 mL) under nitrogenwas treated dropwise with 1N BBr₃ in dichloromethane (2.0 ml, 2.0 mmol).The reaction was allowed to warm to room temperature and stirred for 2h. The resultant suspension was cooled on an ice bath, quenched withsaturated aqueous ammonium chloride (5 ml) and stirred for an additional1 h. The resulting precipitate was filtered and washed with water anddichloromethane, then dissolved in methanol (20 ml), dried overanhydrous MgSO₄, filtered and concentrated in vacuo to affordN-(2-(dimethylamino)ethyl)-2-(3,4,5-trihydroxybenzoyl)-benzofuran-5-carboxamide(NED-2018) as beige solid (93 mg, 97%). ¹H NMR (Methanol-d4): δ 8.4 (d,J=1.8 Hz, 1H), 8.07 (dd, J=8.8, 1.8 Hz, 1H), 7.74 (d, J=8.8 Hz, 1H),7.69 (d, J=1.0 Hz, 1H), 7.19 (s, 2H), 3.80 (t, J=5.8 Hz, 2H), 3.42 (t,J=5.8 Hz, 2H), 3.34 (s, 1H), 3.30 (s, 6H).

N-(2-(diethylamino)ethyl)-2-(3,4,5-trihydroxybenzoyl)-benzofuran-5-carboxamide(NED-2019)

A suspension of2-{[3,4,5-tris(benzyloxy)-phenyl]carbonyl}-1-benzofuran-5-carbonylchloride (137 mg, 0.99 mmol), N,N-diethylethylenediamine (39 mg, 0.33mmol) and potassium carbonate (51.6 mg, 0.53 mmol) in DCM (3 mL) and H₂O(3 mL) was vigorously stirred at room temperature overnight (18 h). Theresulting two layers were separated and the organic layer was washedwith brine, dried over MgSO₄ and concentrated. The residue was purifiedby flash chromatography (10% diethylamine in Ethyl Acetate) to affordN-[2-(diethylamino)ethyl]-2-{[3,4,5-tris(benzyloxy)phenyl]carbonyl}-1-benzofuran-5-carboxamide.

A cooled (−70° C.) stirred solution ofN-[2-(diethylamino)ethyl]-2-{[3,4,5-tris(benzyloxy)phenyl]carbonyl}-1-benzofuran-5-carboxamide(200 mg, 0.29 mmol) in anhydrous dichloromethane (5 mL) under nitrogenwas treated dropwise with 1N BBr3 in dichloromethane (2.0 ml, 2.0 mmol).The reaction was allowed to warm to room temperature and stirred for 2h. The resultant suspension was cooled on an ice bath, quenched withsaturated aqueous ammonium chloride (5 ml) and stirred for an additional1 h. The resulting precipitate was filtered and washed with water anddichloromethane, then dissolved in methanol (20 ml), dried overanhydrous MgSO₄, filtered and concentrated in vacuo to affordN-(2-(diethylamino)ethyl)-2-(3,4,5-trihydroxybenzoyl)-benzofuran-5-carboxamide(NED-2019) as tan solid (65 mg, 54%). ¹H NMR (Methanol-d₄): δ 8.34 (d,J=1.8 Hz, 1H), 8.03 (dd, J=8.8, 1.9 Hz, 1H), 7.75 (d, J=8.7 Hz, 1H),7.68 (d, J=0.9 Hz, 1H), 7.18 (s, 2H), 3.77 (t, J=6.1 Hz, 2H), 3.46-3.31(m, 6H), 1.35 (t, J=7.3 Hz, 6H).

N-(2,4-dimethylphenyl)-2-(3,4,5-trihydroxybenzoyl)benzofuran-5-carboxamide(NED-2021)

An ice cooled stirred solution of2-{[3,4,5-tris(benzyloxy)phenyl]carbonyl}-3a,7a-dihydro-1-benzofuran-5-carboxylicacid (250 mg, 0.43 mmol), 2,4-dimethyl aniline (62 mg, 0.51 mmol) andHOBT hydrate (81.6 mg, 0.53 mmol) in anhydrous DCM (4 mL) under nitrogenwas treated with trimethylamine (0.18 mL, 1.28 mmol) and EDCI (102 mg,0.53 mmol). Allowed reaction to warm to room temperature and stirovernight (18 h). The resulting reaction was concentrated to an oilyresidue, which was purified by flash chromatography (10% diethylamine inEthyl Acetate) to affordN-(2,4-dimethylphenyl)-2-{[3,4,5-tris(benzyloxy)phenyl]carbonyl}-3a,7a-dihydro-1-benzofuran-5-carboxamide.

A cooled (−70° C.) stirred solution ofN-(2,4-dimethylphenyl)-2-{[3,4,5-tris(benzyloxy)phenyl]carbonyl}-1-benzofuran-5-carboxamide(172.3 mg, 0.25 mmol) in anhydrous dichloromethane (5 mL) under nitrogenwas treated dropwise with 1N BBr₃ in dichloromethane (2.0 ml, 2.0 mmol).The reaction was allowed to warm to room temperature and stirred for 2h. The resultant suspension was cooled on an ice bath, quenched withsaturated aqueous ammonium chloride (5 ml) and stirred for an additional1 h. The resulting precipitate was filtered and washed with water anddichloromethane, then dissolved in methanol (20 ml), dried overanhydrous MgSO₄, filtered and concentrated in vacuo to affordN-(2,4-dimethylphenyl)-2-{[3,4,5-tris(benzyloxy)phenyl]carbonyl}-1-benzofuran-5-carboxamide(NED-2021) as light brown solid (100 mg, 96%). ¹H NMR (Methanol-d₄): δ8.62 (d, J=1.9 Hz, 1H), 8.24 (dd, J=8.9, 2.0 Hz, 1H), 7.90 (d, J=8.8 Hz,1H), 7.78 (s, 1H), 7.20 (d, J=14.5 Hz, 3H), 7.05 (s, 1H), 2.52 (s, 3H),2.33 (s, 3H).

(4-isopropylpiperazin-1-yl)(2-(3,4,5-trihydroxybenzoyl)-benzofuran-5-yl)methanone(NED-2022)

A suspension of2-{[3,4,5-tris(benzyloxy)-phenyl]carbonyl}-1-benzofuran-5-carbonylchloride (210 mg, 0.35 mmol), 1-isopropylpiperazine (54 mg, 0.42 mmol)and potassium carbonate (144.4 mg, 1.04 mmol) in DCM (3 mL) and H₂O (3mL) was vigorously stirred at room temperature overnight (18 h). Theresulting two layers were separated and the organic layer was washedwith brine, dried over MgSO4 and concentrated. The residue was purifiedby flash chromatography (10% diethylamine in Ethyl Acetate) to afford1-(propan-2-yl)-4-[(2-{[3,4,5-tris(benzyloxy)phenyl]carbonyl}-1-benzofuran-5-yl)carbonyl]piperazine.

A cooled (−70° C.) stirred solution of1-(propan-2-yl)-4-[(2-{[3,4,5-tris(benzyloxy)phenyl]carbonyl}-1-benzofuran-5-yl)carbonyl]piperazine(153 mg, 0.22 mmol) in anhydrous dichloromethane (5 mL) under nitrogenwas treated dropwise with 1N BBr₃ in dichloromethane (2.0 ml, 2.0 mmol).The reaction was allowed to warm to room temperature and stirred for 2h. The resultant suspension was cooled on an ice bath, quenched withsaturated aqueous ammonium chloride (5 ml) and stirred for an additional1 h. The resulting precipitate was filtered and washed with water anddichloromethane, then dissolved in methanol (20 ml), dried overanhydrous MgSO4, filtered and concentrated in vacuo to afford(4-isopropylpiperazin-1-yl)(2-(3,4,5-trihydroxybenzoyl)-benzofuran-5-yl)methanone(NED-2022) as light brown solid (92 mg, 98%). ¹H NMR (Methanol-d₄): δ7.99 (dd, J=1.7, 0.7 Hz, 1H), 7.77 (d, J=8.6 Hz, 1H), 7.69-7.62 (m, 2H),7.37 (d, J=1.6 Hz, 1H), 7.34-7.29 (m, 1H), 7.18 (s, 2H), 4.61 (s, 1H),3.61-3.23 (m, 8H), 1.39 (d, J=6.7 Hz, 6H).

N-((1-methylpiperidin-4-yl)methyl)-2-(3,4,5-trihydroxybenzoyl)-benzofuran-5-carboxamide(NED-2023)

An ice cooled stirred solution of2-{[3,4,5-tris(benzyloxy)phenyl]carbonyl}-3a,7a-dihydro-1-benzofuran-5-carboxylicacid (200 mg, 0.34 mmol), (1-methyl-4-piperidinyl)-methylamine (52 mg,0.41 mmol) and HOBT hydrate (65 mg, 0.43 mmol) in anhydrous DCM (4 mL)under nitrogen was treated with trimethylamine (0.14 mL, 1.02 mmol) andEDCI (82 mg, 0.43 mmol). Allowed reaction to warm to room temperatureand stir overnight (18 h). The resulting reaction was concentrated to anoily residue, which was purified by flash chromatography (10%diethylamine in Ethyl Acetate) to affordN-[(1-methylpiperidin-4-yl)methyl]-2-[(3,4,5-trihydroxyphenyl)carbonyl]-1-benzofuran-5-carboxamide.

A cooled (−70° C.) stirred solution ofN-[(1-methylpiperidin-4-yl)methyl]-2-[(3,4,5-trihydroxyphenyl)carbonyl]-1-benzofuran-5-carboxamide(139 mg, 0.20 mmol) in anhydrous dichloromethane (5 mL) under nitrogenwas treated dropwise with 1N BBr3 in dichloromethane (2.0 ml, 2.0 mmol).The reaction was allowed to warm to room temperature and stirred for 2h. The resultant suspension was cooled on an ice bath, quenched withsaturated aqueous ammonium chloride (5 ml) and stirred for an additional1 h. The resulting precipitate was filtered and washed with water anddichloromethane, then dissolved in methanol (20 ml), dried overanhydrous MgSO₄, filtered and concentrated in vacuo to affordN-((1-methylpiperidin-4-yl)methyl)-2-(3,4,5-trihydroxybenzoyl)-benzofuran-5-carboxamide(NED-2023) as light brown solid (54 mg, 64%). ¹H NMR (Methanol-d₄): δ8.29 (d, J=1.8 Hz, 1H), 7.98 (dd, J=8.8, 1.9 Hz, 1H), 7.70 (d, J=8.8 Hz,1H), 7.66 (d, J=0.9 Hz, 1H), 7.18 (s, 2H), 3.52 (d, J=12.1 Hz, 2H), 3.37(d, J=6.5 Hz, 2H), 2.99 (s, 1H), 2.85 (s, 3H), 2.04 (d, J=14.8 Hz, 2H),1.55 (d, J=13.5 Hz, 2H).

Preparation of Aurones(Z)-2-(3,4,5-Trihydroxybenzylidene)benzofuran-3(2H)-one (APY-024)

A stirred solution of3,4,5-tris((tert-butyldimethylsilyl)oxy)benzaldehyde (Horenstein andNakanishi, 1989, J. Am. Chem. Soc, 111:6242-6246)(1.84 g, 3.7 mmol) andbenzofuran-3(2H)-one (0.55 g, 4.1 mmol) in dichloromethane (20 mL) wastreated with neutral alumina (14 g) and stirred for 1 hour, then dilutedwith methanol (10 mL) and filtered. The solid was rinsed with 2:1DCM/methanol until color wasn't seen in the filtrate and no more productdetected by TLC, and the filtrate was concentrated in vacuo. Theresidual solid was recrystallized from acetonitrile to afford 1.66 g(73%) of trisilylated intermediate as a bright yellow solid, which wascarried through without characterization. An ice-cooled (3° C.) stirredsolution of this compound (1.594 g, 2.6 mmol) in anhydrous THF (25 mL)under argon was treated with 1N tetrabutylammonium fluoride/THF (9.6 mL,9.6 mmol) and stirred at 3° C. for 30 minutes and at room temperaturefor 1 hour. Saturated aqueous ammonium chloride (50 mL) was added,followed by adjustment to pH˜3 with 2N HCl (whereupon the colorlightened to yellow orange). The mixture was extracted with ethylacetate (150 mL, then 2×50 mL) and the combined organic solution waswashed with water and brine (50 mL each), dried (MgSO4) and concentratedin vacuo to a yellow-orange solid. This was triturated from acetonitrileto afford 692 mg (98.5%) of(Z)-2-(3,4,5-trihydroxybenzyl-idine)benzo-furan-3(2H)-one as an orangesolid (FIG. 12 ). ¹H NMR (d6-DMSO): δ 9.17 (br s, 3H), 7.75 (m, 2H),7.46 (d, J=8.5 Hz, 1H), 7.27 (t, J=7.5 Hz, 1H), 6.98 (s, 2H), 6.89 (s,1H). ¹³C NMR (d6-DMSO): δ 183.31, 165.24, 146.48, 145.13, 137.44,137.34, 124.47, 124.01, 122.37, 121.73, 114.82, 113.28, 111.68. LCMSm/z: [M−H]⁻ 269.8 (100%).

(Z)-2-(3,4-Dimethoxybenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one(APY-001)

A stirred mixture of 4,6-dihydroxybenzofuran-3(2H)-one (Haudecoeur etal., 2011, Journal of medicinal chemistry, 54:5395-5402) (166 mg, 1.0mmol) and 3,4-dimethoxybenzaldehyde (249 mg, 1.5 mmol) in methanol (15mL) was treated with 50% aqueous potassium hydroxide (1.5 mL) and heatedto reflux for 5 hours, then cooled to room temperature. Methanol wasremoved in vacuo and replaced with water (30 mL), and the solution waswashed with ether (2×20 mL, decanted from mixture). The solution wasacidified to pH˜4 with concentrated HCl, and the resultant suspensionwas filtered, washed several times with water, and the solid dried invacuo overnight. Recrystallization from acetonitrile afforded 232 mg(74%) of(Z)-2-(3,4-dimethoxybenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one as ayellow solid (FIG. 13 ). ¹H NMR (d6-DMSO): δ 10.90 (br s, 1H), 10.86 (brs, 1H), 7.52 (m, 2H), 7.07 (d, J=7 Hz, 1H), 6.59 (s, 1H), 6.24 (d, J=2Hz, 1H), 6.11 (d, J=2 Hz, 1H), 3.83 (s, 3H), 3.82 (s, 3H). ¹³C NMR(d6-DMSO): δ 179.32, 167.93, 167.52, 158.59, 150.29, 149.06, 146.91,125.43, 124.80, 114.32, 112.34, 109.18, 103.06, 98.07, 90.92, 55.93,55.88. LCMS m/z: [M−H]⁻ 313.1 (30%). LCMS m/z: [M+H]⁺ 315.2 (100%).

(Z)-2-(3,4-Dihydroxybenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one(APY-007)

An ice-cooled stirred suspension of(Z)-2-(3,4-dimethoxybenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one (126mg, 0.40 mmol) in anhydrous methylene chloride (3 mL) under argon wastreated dropwise with 1N boron tribromide/dichloromethane (5 mL, 5mmol), then allowed to reach room temperature overnight. The dark redmixture was added to ice (40 mL) and extracted with ethyl acetate (4×40mL). The combined organic solution was washed with water (3×75 mL),brine (75 mL), dried (MgSO₄), and concentrated in vacuo. The residualsolid was triturated from acetonitrile to afford 52 mg (45%) of(Z)-2-(3,4-dihydroxybenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one as ayellow solid (FIG. 14 ). ¹H NMR (d6-DMSO): δ 10.25-11.25 (br s, 2H),8.75-10.00 (br s, 2H), 7.44 (d, J=2 Hz, 1H), 7.23 (dd, J=8 Hz, 2 Hz,1H), 6.87 (d, J=8 Hz, 1H), 6.49 (s, 1H), 6.21 (d, J=2 Hz, 1H), 6.11 (d,J=2 Hz, 1H). ¹³C NMR (d6-DMSO): δ 179.31, 167.87, 167.51, 158.64,147.77, 146.29, 145.79, 124.22, 124.03, 117.91, 116.30, 109.79, 103.16,98.04, 90.61. LCMS m/z: [M−H]⁻ 284.8 (100%). LCMS m/z: [M+H]⁺ 286.8(100%).

(Z)-2-(3,5-Dihydroxybenzylidene)-4,6-dihydroxybenzofuran-3(2H)-one(APY-014)

A stirred mixture of 4,6-dihydroxybenzofuran-3(2H)-one (31) (166 mg, 1.0mmol) and 3,5-dimethoxybenzaldehyde (249 mg, 1.5 mmol) in methanol (15mL) was treated with 50% aqueous potassium hydroxide (1.5 mL) and heatedto reflux for 5 hours, then cooled to room temperature. The solution wasconcentrated in vacuo and the residue dissolved in water (15 mL) andacidified to pH˜2 with HCl. The resultant suspension was cooled on anice bath, filtered, and the solid washed several times with water anddried in vacuo. Trituration from cold acetonitrile afforded 240 mg (76%)of penultimate intermediate as a pale beige solid, carried forwardwithout characterization. A cooled (−70° C.) suspension of this compound(235 mg, 0.75 mmol) in anhydrous dichloromethane (5 mL) under argon wastreated dropwise via syringe with 1N boron tribromide/dichloromethane (5mL) and allowed to reach 0° C. over 1.5 hours, then maintained on an icebath and allowed to reach room temperature overnight (16 hours). Thereaction mixture was poured onto ice, stirred several minutes, filtered,and the solid rinsed several times with water and air dried. The solidwas dissolved in dichloromethane containing a little methanol, loadedonto a short column of silica gel (˜25 cc) and eluted with 10%methanol/ethyl acetate to afford a dark brown solid. This was trituratedfrom cold ether to afford 90 mg (42%) of(Z)-2-(3,5-dihydroxybenz-ylidene)-4,6-dihydroxybenzofuran-3(2H)-one as abrown powder (FIG. 15 ). ¹H NMR (d6-DMSO): δ 10.25-11.50 (br s, 2H),9.15-9.75 (br s, 2H), 6.77 (d, J=2 Hz, 2H), 6.37 (s, 1H), 6.28 (t, J=2Hz, 1H), 6.17 (d, J=2 Hz, 1H), 6.08 (d, J=2 Hz, 1H). ¹³C NMR (d6-DMSO):δ 179.41, 168.13, 167.87, 158.85, 147.88, 133.97, 109.21, 109.11,105.95, 104.38, 102.90, 98.18, LCMS m/z: EM-fir 284.8 (100%). LCMS m/z:[M+H]⁺ 286.8 (100%).

(Z)-4-Hydroxy-2-(3,4,5-trihydroxybenzylidene)benzofuran-3(2H)-one(APY-019)

A stirred mixture of 4-hydroxybenzofuran-3(2H)-one (Singh et al., 2010,Journal of medicinal chemistry, 53:18-36) (150 mg, 1.0 mmol) and3,4,5-trimethoxybenzaldehyde (294 mg, 1.5 mmol) in methanol (15 mL) wastreated with 50% aqueous potassium hydroxide (1.5 mL) and heated toreflux for 5 hours, then cooled to room temperature and concentrated invacuo. Water (15 mL) was added, the mixture cooled on an ice bath, andacidified with concentrated HCl to pH˜3 (yellow precipitate formed).Stirring on ice was continued for several minutes, then the suspensionwas filtered, rinsed several times with cold water, and dried in vacuo.The solid was recrystallized from acetonitrile and dried to afford 259mg (79%) of penultimate intermediate as a yellow solid, carried forwardwithout characterization. A cooled (−65° C.) stirred suspension of thiscompound (213 mg, 0.65 mmol) in anhydrous dichloromethane (5 mL) underargon was treated with 1N BBr3/dichloromethane (5 mL, 5 mmol) at a rateto keep pot temp below −50° C., then allowed to reach 0° C. over 1.5hours. The mixture was placed on an ice bath and allowed to reach roomtemperature overnight. The reaction was quenched by addition to ice andstirring, and the aqueous suspension was filtered and the solid rinsedseveral times with water and partially air dried. Trituration fromacetonitrile afforded 151 mg (81%) of(Z)-4-hydroxy-2-(3,4,5-trihydroxybenzylidene)benzofuran-3(2H)-one as ayellow-brown solid (FIG. 16 ). ¹H NMR (d6-DMSO): δ 11.01 (br s, 1H),9.20 (br s, 2H), 8.90 (br s, 1H), 7.52 (t, J=7 Hz, 1H), 6.96 (s, 2H),6.80 (d, J=8 Hz, 1H), 6.63 (d, J=8 Hz, 1H), 6.52 (s, 1H). ¹³C NMR(d6-DMSO): δ 181.35, 166.02, 157.29, 146.41, 145.26, 138.47, 136.64,122.61, 112.31, 111.16, 110.65, 109.81, 102.58. LCMS m/z: [M−H]⁻ 285.6(100%).

Preparation of Benzohydrazides 3,4,5-trihydroxybenzohydrazide (NED-2047)

A solution of methyl gallate (4 g, 21.7 mmol) in ethanol (25 mL) washydrazine hydrate (4.35 g, 86.9 mmol) and heated to a gentle reflux.After stirring for 18 h, the reaction mixture was cooled to roomtemperature, and the precipitated product was filtered and sequentiallywashed with water and ethanol and dried in a vacuum oven to give thetitle compound (3.85 g, 96%) as a white solid. 1H NMR (DMSO-D6) 9.29 (s,1H), 8.97 (br s, 2H), 8.60 (br s, 1H), 6.74 (s, 2H), 4.28 (s, 2H).LC-MS: m/z=185 [M+].

N′-(2,4-dimethoxybenzylidene)-3,4,5-trihydroxybenzohydrazide (NED-2036)

A suspension of 3,4,5-trihydroxybenzohydrazide (367 mg, 1.99 mmol;NED-2047) in ethanol (6 mL) was treated with 2,4-dimethoxybenzaldehyde(300 mg, 1.81 mmol) and heated to a gentle reflux for 16 h. Aftercooling to room temperature, the precipitated product was washed withethanol (2×10 mL) and hexanes (2×5 mL). The precipitate was dried in avacuum oven for 24 h to give the title compound as a white solid (155mg, 26%). 1H NMR (DMSO-D6) 11.38 (s, 1 H), 9.30 (s, 1H), 8.98 (br s,2H), 8.79 (s, 1H), 8.63 (s, 1H), 7.87 (d, J=8.5 Hz, 1H), 7.73 (d, J=9.1Hz, 1H), 6.88 (s, 1H), 6.75 (s, 1H), 6.63-6.57 (m, 4H), 3.84-3.78 (m,12H). LC-MS: m/z=333 [M+].

(E)-3,4,5-trihydroxy-N′-((2-hydroxynaphthalen-1-yl)methylene)benzohydrazide(NED-2037)

A suspension of 3,4,5-trihydroxybenzohydrazide (300 mg, 1.63 mmol;NED-2047) in methanol (10 mL) was treated with2-hydroxy-1-naphthaldehyde (297 mg, 1.72 mmol) and heated to a gentlereflux for 16 h. After cooling to room temperature, the precipitatedproduct was washed with methanol (2×10 mL). The precipitate was dried ina vacuum oven for 24 h to give the title compound as a white solid (205mg, 37%). 1H NMR (DMSO-D6) 9.38 (s, 1H), 8.12 (d, J=8.6 Hz, 1H),7.83-7.78 (m, 2H), 7.55-7.50 (m, 1H), 7.37-7.33 (m, 1H), 7.16 (d, J=9.0Hz, 1H), 7.00 (s, 2H). LC-MS: m/z=339 [M+].

(E)-N′-((5-(4-bromophenyl)furan-2-yl)methylene)-3,4,5-trihydroxybenzohydrazide(NED-2038)

A suspension of 3,4,5-trihydroxybenzohydrazide (400 mg, 2.17 mmol;NED-2047) in methanol (10 mL) was treated with 5-(4-bromophenyl)furfural(545 mg, 2.17 mmol) and heated to a gentle reflux for 16 hours. Aftercooling to room temperature, the precipitated product was washed withmethanol (2×10 mL). The precipitate was dried in a vacuum oven for 24hours to give the title compound as a white solid (385 mg, 42%). 1H NMR(DMSO-D6) 11.53 (s, 1H), 9.13 (bs, 1H), 8.30 (s, 1H), 7.71 (d, J=8.6 Hz,2H), 7.63 (d, J=8.6 Hz, 2H), 7.16 (d, J=3.6 Hz, 2H), 6.97 (d, J=3.6 Hz,2H), 6.88 (s, 2H). LC-MS: m/z=418,420 [M+].

(E)-3,4,5-trihydroxy-N′-(3,4,5-trimethoxybenzylidene)benzohydrazide(NED-2039)

A suspension of 3,4,5-trihydroxybenzohydrazide (300 mg, 1.63 mmol;NED-2047) in methanol (12 mL) was treated with3,4,5-trimethoxybenzaldehyde (352 mg, 1.79 mmol) and heated to a gentlereflux for 16 h. After cooling to room temperature, the precipitatedproduct was washed with methanol (2×10 mL) and ethylacetate/dichloromethane (1×10 mL, 1:1). The precipitate was dried in avacuum oven for 24 hours to give the title compound as a white solid(165 mg, 28%). 1H NMR (DMSO-D6) 11.51 (s, 1H), 9.14 (bs, 2H), 8.82 (bs,1H), 8.31 (s, 1H), 6.95 (s, 2H), 6.89 (s, 2H), 3.82 (s, 6H), 3.68 (s,3H). LC-MS: m/z=363 [M+].

(E)-N′-(4-(dimethylamino)benzylidene)-3,4,5-trihydroxybenzohydrazide(NED-2048)

A suspension of 3,4,5-trihydroxybenzohydrazide (300 mg, 1.63 mmol;NED-2047) in methanol (12 mL) was treated with4-dimethylaminobenzaldehyde (267 mg, 1.79 mmol) and heated to a gentlereflux for 16 hours. After cooling to room temperature, the precipitatedproduct was washed with methanol (2×10 mL) and ethylacetate/dichloromethane (1×10 mL, 1:1). The precipitate was dried in avacuum oven for 24 hours to give the title compound as a white solid(225 mg, 44%). 1H NMR (DMSO-D6) 11.21 (s, 1H), 9.08 (bs, 2H), 8.77 (bs,1H), 8.22 (s, 1H), 7.46 (d, J=8.5 Hz, 2H), 6.85 (s, 2H), 6.72 (d, J=8.6Hz, 2H), 2.93 (s, 6H).

(E)-3,4,5-trihydroxy-N′-(naphthalen-1-ylmethylene)benzohydrazide(NED-2134)

A suspension of 3,4,5-trihydroxybenzohydrazide (300 mg, 1.63 mmol;NED-2047) in methanol (12 mL) was treated with 1-naphthaldehyde (352 mg,1.79 mmol) and heated to a gentle reflux for 16 hours. After cooling toroom temperature, the precipitated product was washed with methanol(2×10 mL) and ethyl acetate/dichloromethane (1×10 mL, 1:1). Theprecipitate was dried in a vacuum oven for 24 hours to give the titlecompound as a white solid (165 mg, 28%). 1H NMR (DMSO-D6) 11.59 (s, 1H),9.17 (s, 2H), 9.05 (s, 1H), 8.85 (s, 1H), 8.80 (d, J=8.5 Hz, 1H), 7.98(d, J=8.1 Hz, 2H), 7.87 (d, J=7.2 Hz, 1H), 7.63-7.55 (m, 3H), 6.94 (s,2H), LC-MS: m/z=323 [M+].

(E)-3,4,5-trihydroxy-N′-(naphthalen-2-ylmethylene)benzohydrazide(NED-2135)

A suspension of 3,4,5-trihydroxybenzohydrazide (280 mg, 1.52 mmol;NED-2047) in methanol (10 mL) was treated with 2-naphthaldehyde (250 mg,1.60 mmol) and heated to a gentle reflux for 16 hours. After cooling toroom temperature, the precipitated product was washed with methanol(2×10 mL) and ethyl acetate/methanol (1×10 mL, 1:1). The precipitate wasdried in a vacuum oven for 24 hours to give the title compound as awhite solid (455 mg, 93%). 1H NMR (DMSO-D6) 11.63 (s, 1H), 9.17 (s, 2H),9.05 (s, 1H), 8.84 (s, 1H), 8.55 (s, 1H), 8.08 (s, 1H), 8.00-7.91 (m,4H), 7.56-7.53 (m, 2H), 6.93 (s, 2H). LC-MS: m/z=323 [M+]; 345 [M+Na].

(E)-3,4,5-trihydroxy-N′46-methoxynaphthalen-2-yl)methylene)benzohydrazide(NED-2136)

A suspension of 3,4,5-trihydroxybenzohydrazide (250 mg, 1.36 mmol;NED-2047) in methanol (10 mL) was treated with6-methoxy-2-naphthaldehyde (266 mg, 1.43 mmol) and heated to a gentlereflux for 16 hours. After cooling to room temperature, the precipitatedproduct was washed with methanol (2×10 mL) and ethyl acetate/methanol(1×10 mL, 1:1). The precipitate was dried in a vacuum oven for 24 hoursto give the title compound as a white solid (465 mg, 96%). 1H NMR(DMSO-D6) 11.54 (s, 1H), 9.15 (s, 2H), 8.82 (s, 1H), 8.48 (s, 1H), 7.99(s, 1H), 7.89-7.81 (m, 3H), 7.33 (d, J=2.5 Hz, 1H), 7.19-7.16 (m, 1H),6.90 (s, 2H). LC-MS: m/z=353 [M+].

(E)-3,4,5-trihydroxy-N′-((2-methoxynaphthalen-1-yl)methylene)benzohydrazide(NED-2173)

A suspension of 3,4,5-trihydroxybenzohydrazide (220 mg, 1.12 mmol;NED-2047) in methanol (10 mL) was treated with2-methoxy-1-naphthaldehyde (220 mg, 1.18 mmol) and heated to a gentlereflux for 48 hours. After cooling to room temperature, the precipitatedproduct was washed with methanol (2×10 mL). The precipitate was dried ina vacuum oven for 24 hours to give the title compound as a white solid(282 mg, 71%). 1H NMR (DMSO-D6) 11.60 (s, 1H), 9.38 (d, J=8.7 Hz, 1H),9.13 (s, 2H), 9.10 (s, 1H), 8.01 (d, J=9.1 Hz, 1H), 7.88 (d, J=8.0 Hz,1H), 7.56-7.53 (m, 1H), 7.49 (d, J=9.2 Hz, 1H), 7.42-7.38 (m, 1H), 6.96(s, 2H). LC-MS: m/z=353 [M+].

(E)-3,4,5-trihydroxy-N′-((2-(prop-2-yn-1-yloxy)naphthalen-1-yl)methylene)benzohydrazide(NED-2174)

2-hydroxy-1-naphthaldehyde (350 mg, 2.03 mmol) was dissolved inanhydrous DMF (2 mL). Anhydrous K₂CO₃ (618 mg, 4.47 mmol) was added tothis solution at room temperature, stirred for 20 minutes, and thencharged with propargyl bromide (544 mg, 3.66 mmol). The resultingmixture was stirred at room temperature for 72 hours. After thecompletion of the reaction, ethyl acetate (30 mL) was added, the organicphase was washed with water (3×50 mL), dried over MgSO4, and filtered.Removal of solvent gave oily residue which was purified over silica gelflash chromatography (Eluent: 100% hexanes to 20% ethyl acetate inhexanes). 2-(prop-2-yn-1-yloxy)naphthalene-1-carbaldehyde was isolatedas a clear oil and fraction one (410 mg, 96%). 1H NMR (CDCl₃) 10.91 (s,1H), 9.29-9.26 (m, 1H), 8.08 (d, J=9.1 Hz, 1H), 7.81-7.78 (m, 1H),7.66-7.62 (m, 1H), 7.47-7.43 (m, 1H), 7.39 (d, J=9.2 Hz, 1H), 4.96 (s,2H), 2.58 (s, 1H).

A suspension of 3,4,5-trihydroxybenzohydrazide (216 mg, 1.17 mmol;NED-2047) in methanol (10 mL) was treated with2-(prop-2-yn-1-yloxy)naphthalene-1-carbaldehyde (260 mg, 1.24 mmol) andheated to a gentle reflux for 16 hours. After cooling to roomtemperature, the precipitated product was washed with methanol (2×10mL). The precipitate was dried in a vacuum oven for 24 hours to give thetitle compound as a white solid (270 mg, 61%). 1H NMR (DMSO-D6) 11.67(s, 1H), 9.37 (d, J=8.7 Hz, 1H), 9.13 (bs, 2H), 9.07 (s, 1H), 8.74 (bs,1H), 8.00 (d, J=9.1 Hz, 1H), 7.88 (d, J=8.1 Hz, 1H), 7.55-7.51 (m, 2H),7.44-7.41 (m, 1H), 6.95 (s, 2H), 3.62 (s, 1H). LC-MS: m/z=377 [M+].

(E)-3,4,5-trihydroxy-N′-((2-(pyridin-2-ylmethoxy)naphthalen-1-yl)methylene)benzohydrazide(NED-2175)

2-hydroxy-1-naphthaldehyde (350 mg, 2.03 mmol) was dissolved inanhydrous DMF (2 mL). Anhydrous K₂CO₃ (1.01 g, 7.32 mmol) was added tothis solution at room temperature, stirred for 20 minutes, and thencharged with 2-(Bromomethyl)pyridine hydrobromide (925 mg, 3.66 mmol).The resulting mixture was stirred at room temperature for 72 hours.After the completion of the reaction, ethyl acetate (30 mL) was added,the organic phase was washed with water (3×50 mL), dried over MgSO4, andfiltered. Removal of solvent gave oily residue which was purified oversilica gel flash chromatography (Eluent: 100% hexanes to 65% ethylacetate in hexanes). 2-(pyridin-2-ylmethoxy)naphthalene-1-carbaldehydewas isolated as a clear oil and fraction one (415 mg, 78%). 1H NMR(CDCl₃) 11.07 (s, 1H), 9.29 (m, 1H), 8.63 (m, 1H), 8.04 (d, J=9.1 Hz,1H), 7.79-7.73 (m, 2H), 7.66-7.61 (m, 1H), 7.56 (d, J=7.8 Hz, 1H),7.46-7.41 (m, 1H), 7.34 (d, J=7.8 Hz, 1H), 5.47 (s, 2H).

A suspension of 3,4,5-trihydroxybenzohydrazide (200 mg, 1.09 mmol;NED-2047) in methanol (10 mL) was treated with2-(pyridin-2-ylmethoxy)naphthalene-1-carbaldehyde (301 mg, 1.14 mmol)and heated to a gentle reflux for 16 hours. After cooling to roomtemperature, the precipitated product was washed with methanol (2×10mL). The precipitate was dried in a vacuum oven for 24 hours to give thetitle compound as a white solid (180 mg, 39%). 1H NMR (DMSO-D6) 11.68(s, 1H), 9.37 (d, J=8.8 Hz, 1H), 9.19 (s, 1H), 9.14 (s, 2H), 8.80 (s,1H), 8.57 (d, J=5.0 Hz, 1H), 7.96 (d, J=9.1 Hz, 1H), 7.87-7.83 (m, 2H),7.66 (d, J=7.8 Hz, 1H), 7.58-7.52 (m, 1H), 7.42-7.39 (m, 1H), 7.35-7.32(m, 1H), 6.94 (s, 2H), 5.42 (s, 2H). LC-MS: m/z=430 [M+].

(E)-N′-(cyclohexylmethylene)-3,4,5-trihydroxybenzohydrazide (NED-2251)

A suspension of 3,4,5-trihydroxybenzohydrazide (200 mg, 1.09 mmol;NED-2047) in methanol (8 mL) was treated with cyclohexanecarboxaldehyde(128 mg, 1.14 mmol) and heated to a gentle reflux for 18 hours. Aftercooling to room temperature, volatiles were removed. chloroform and 5 mLdichloromethane were added to the oily residue following concentrationwhich resulted in precipitation of a white solid. The precipitate wasfiltered, washed with more dichloromethane (5 mL), diethyl ether (2×5mL), collected, and dried in a vacuum oven for 24 hours to give thetitle compound as a white solid (265 mg, 88%). 1H NMR (DMSO-D6) 11.00(s, 1H), 9.06 (s, 2H), 8.72 (s, 1H), 7.56 (d, J=5.4 Hz, 1H), 6.79 (s,2H), 2.17 (bs, 1H), 1.73-1.66 (m, 4H), 1.60-1.57 (m, 1H), 1.27-1.13 (m,5H). LC-MS: m/z=279 [M+].

Preparation of Additional CompoundsN-[(3,4-Dimethoxyphenyl)methyl]-3,4,5-trihydroxybenzamide (APY-077)

An ice cooled (3° C.) stirred solution of 3,4,5-triacetoxybenzoylchloride (Bian et al., 2013, Bioorg Med Chem, 21:5442-5450) (315 mg, 1.0mmol) in dichloromethane (5 mL) under nitrogen was treated withtriethylamine (0.28 mL) then dropwise with 3,4-dimethoxybenzylamine (167mg, 1.0 mmol) in dichloromethane (3 mL), warmed to room temperature,stirred overnight, and concentrated in vacuo. The residue was dissolvedin dichloromethane and loaded onto a silica gel column (˜80 cc) andeluted with 3:2 dichloromethane/ethyl acetate to afford 390 mg (88%) ofpenultimate intermediate as a white foam. A stirred suspension of thiscompound (381 mg, 0.855 mmol) in ethanol (10 mL) under nitrogen wastreated with hydrazine hydrate (0.175 mL, 3.6 mmol) and stirred at roomtemperature for 2 hours, then concentrated in vacuo. The residual solidwas stirred in water (12 mL) for 15 minutes, filtered, and the solidrinsed with water, collected and dried in vacuo to afford 238 mg (87%)of N-[(3,4-dimethoxyphenyl)methyl]-3,4,5-trihydroxybenzamide as a whitepowder. ¹H NMR (d6-acetone): δ 8.37 (br s, 2H), 8.00 (br s, 2H), 7.00(m, 3H), 6.86 (m, 2H), 4.44 (s, 2H), 3.76 (s, 6H). ¹³C NMR (d6-acetone):δ 166.42, 149.34, 148.44, 145.54, 145.48, 136.04, 119.77, 111.90,111.84, 106.88, 106.85, 55.30, 55.20, 42.73. LCMS m/z: [M−H]⁻ 317.6(100%). LCMS m/z: [M+H]⁺ 319.6 (100%).

2-(4-Bromophenyl)-4-[3,4,5-tris(benzyloxy)phenyl]-1,3-thiazole(Intermediate 2)

A stirred mixture of 2-bromo-1-(3,4,5-tris(benzyloxy)phenyl)ethan-1-one(29) (1.3 g, 2.5 mmol) and 4-bromobenzene-1-carbothioamide (0.54 g, 2.5mmol) in ethanol (25 mL) under nitrogen was heated to 80° C. for 4hours, then cooled to room temperature. The suspension was filtered andthe solid rinsed with ethanol, collected and dried in vacuo to afford1.29 g (81%) of2-(4-bromophenyl)-4-[3,4,5-tris(benzyloxy)phenyl]-1,3-thiazole as avoluminous white solid (FIG. 18 ). ¹H NMR (d6-acetone): δ 7.89 (d, J=9Hz, 2H), 7.59 (d, J=9 Hz, 2H), 7.48 (m, 4H), 7.43 (m, 2H), 7.25-7.40 (m,12H), 5.20 (s, 4H), 5.11 (s, 2H). ¹³C NMR (d6-acetone): δ 166.45,156.08, 138.81, 137.73, 137.10, 132.49, 132.08, 129.89, 128.63, 128.50,128.15, 128.00, 127.91, 127.83, 127.54, 127.53, 124.34, 112.49, 106.53,75.25, 71.44. LCMS m/z: [M−H]⁻ 317.6 (100%). LCMS m/z: [M+H]⁺ 635.3(100%).

N-Butyl-4-{4-[4-(3,4,5-trihydroxyphenyl)-1,3-thiazol-2-yl]phenyl}benzene-1-sulfonamide(APY-093)

A stirred mixture of2-(4-bromophenyl)-4-[3,4,5-tris(benzyloxy)phenyl]-1,3-thiazole (254 mg,0.40 mmol), [4-(butylsulfamoyl)phenyl]boronic acid (144 mg, 0.56 mmol)and potassium carbonate (221 mg, 1.6 mmol) in 9:1 dioxane/water (4 mL)in a pressure tube was degassed over 20 minutes with argon, then treatedwith 1,1-bis(diphenylphosphino)ferrocene dichloropalladium (II) (33 mg,0.045 mmol). The tube was capped, and heated to 90° C. for 18 hours,cooled to room temperature and diluted with DCM (12 mL). The mixture wascombined with water (10 mL) and separated. The aqueous solution wasextracted with DCM (8 mL) and the combined organic solution was washedwith water (10 mL), dried (MgSO4) and filtered and concentrated invacuo. The residue was dissolved in DCM and loaded onto a silica gelcolumn (˜80 cc) and eluted with DCM, then 2% EtOAc/dichloromethane toafford 299 mg (97%) of penultimate intermediate as a pale yellow solid.A cooled (−70° C.) stirred solution of this compound (260 mg, 0.339mmol) in anhydrous DCM (6 mL) was subjected to Procedure A with 1NBBr3/DCM (2.7 mL, 2.7 mmol), and the residual solid was triturated fromDCM, then dissolved in 9:1 DCM/methanol and added to a silica gel column(˜90 cc) and eluted with 5%, then 10% methanol/DCM. The solid productwas triturated again from DCM to afford 110 mg (65%) ofN-butyl-4-{4-[4-(3,4,5-trihydroxyphenyl)-1,3-thiazol-2-yl]phenyl}benzene-1-sulfonamideas a pale tan solid (FIG. 19 ). ¹H NMR (d6-acetone): δ 8.17 (d, J=8 Hz,2H), 7.96 (m, 4H), 7.89 (d, J=8 Hz, 2H), 7.75-8.00 (br s, 3H), 7.68 (s,1H), 7.19 (m, 2H), 6.47 (t, J=6 Hz, 1H), 2.95 (q, J=7 Hz, 2H), 1.48 (m,2H), 1.33 (m, 2H), 0.85 (t, J=7 Hz, 3H). ¹³C NMR (d6-acetone): δ 165.96,156.67, 145.87, 143.57, 140.64, 140.33, 133.75, 133.46, 127.82, 127.56,127.38, 126.86, 126.02, 111.47, 105.79, 42.75, 31.53, 19.49, 12.97. LCMSm/z: [M−H]⁻ 494.5 (100%). LCMS m/z: [M+H]⁺ 496.8 (100%).

[3-Fluoro-4-({5-[4-(hydroxymethyl)phenyl]-1-benzofuran-2-yl}carbonyl)phenyl]boronicacid (APY-098)

A stirred suspension of potassium carbonate (207 mg, 1.5 mmol) in methylethyl ketone (1 mL) under nitrogen was treated with a solution of2-hydroxy-5-[4-(hydroxymethyl)phenyl]benzaldehyde (228 mg, 1 mmol) in2-butanone (1 mL) and stirred 15 minutes. A solution of[4-(2-bromoacetyl)-3-fluorophenyl]boronic acid (261 g, 1 mmol) in2-butanone (3 mL) was added, and the mixture heated to 50° C. for 20hours, cooled to room temperature, and diluted with water (20 mL). Theaqueous suspension was stirred a few minutes, filtered, and the filtercake rinsed with water and air-dried. The solid was triturated fromacetonitrile and dried in vacuo to afford 251 mg (64%) of[3-fluoro-4-({5-[4-(hydroxymethyl)phenyl]-1-benzofuran-2-yl}carbonyl)phenyl]boronicacid as a very pale yellow powder (FIG. 20 ). ¹H NMR (d6-DMSO): δ 8.06(m, 1H), 7.45-7.95 (m, 5H), 7.35 (m, 3H), 5.60-6.00 (m, 1H), 5.20 (m,1H), 4.50 (m, 2H). ¹³C NMR (d6-DMSO): δ 155.49, 142.22, 138.65, 127.96,127.53, 127.47, 127.15, 126.35, 121.99, 118.44, 113.10, 63.02, 60.21,40.46, 21.21, 19.00, 14.53, 1.60. LCMS m/z: [M+H]⁺ 391.4 (80%).

Synthesis ofN-(2-(pyrrolidin-1-yl)ethyl)-2-(3,4,5-trihydroxybenzoyl)benzofuran-5-carboxamide(NED-2020)

Potassium carbonate (1.1 g, 7.78 mmol) was added to a solution of5-(methoxycarbonyl)salicylaldehyde (935 mg, 5.19 mmol) in 2-butanone (30ml) and the resulting suspension was stirred at room temperature undernitrogen for 30 minutes. A solution of2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone (Reddy et al., 2017, BioorgMed Chem Lett, 27:1379-1384)¹ (1.5 g, 5.19 mmol) in 20 ml of 2-butanonewas added to the above mixture. The reaction was heated to 50° C.,stirred for 18 hours and cooled to room temperature. Water (150 ml) wasadded and the suspension was stirred for 10 minutes, filtered and rinsedwith water to afford intermediate 1 as a white voluminous solid (1.5 g,78%).

To a suspension of intermediate 1 (1 g, 2.70 mmol) in methanol (20 ml)was added dropwise 20% aqueous NaOH (5 ml), and stirring continued at60° C. for 4 hours. After cooling to room temperature, the suspensionwas acidified to pH 3 with 2N HCl and a light yellow solid formed, whichwas collected by filtration, rinsed with water, and air dried to affordintermediate 2 (850 mg, 88%).

Intermediate 2 (500 mg, 1.40 mmol), 2-pyrrolidin-1-ylethanamine (320 mg,2.80 mmol) and HOBt hydrate (322 mg, 2.11 mmol) were combined indichloromethane (30 ml) under nitrogen, and the suspension was cooled onan ice bath and treated with triethylamine (0.58 mL, 4.21 mmol) and EDChydrochloride (403 mg, 2.11 mmol). The solution was slowly warmed toroom temperature and stirred overnight, concentrated in vacuo, and theresidue dissolved in ethyl acetate (100 ml) and washed with water (2×100ml) and brine (100 ml). The organic solution was dried (MgSO4) andconcentrated in vacuo to afford a brown oil, which was purified bycolumn chromatography on silica gel (eluted with 10% diethylamine/ethylacetate) to yield intermediate 3 as white solid (460 mg, 72%).

A cooled (−70° C.) stirred solution of intermediate 3 (400 mg, 0.88mmol) in anhydrous dichloromethane (15 ml) under nitrogen was treateddropwise with 1N BBr₃/DCM (7.0 ml, 7.0 mmol). The reaction was allowedto warm to room temperature and stirred for 2 hours. The resultant darkorange suspension was cooled on an ice bath, quenched with saturatedaqueous ammonium chloride (5 ml) and stirred for 1 hour. The precipitatewas filtered and the solid washed with water (20 ml) and dichloromethane(20 ml), then dried, filtered and concentrated in vacuo to affordproduct NED-2020 as bright yellow solid (200 mg, 55%) (FIG. 21 ). ¹H NMR(CD₃OD): δ 8.37 (d, J=1.8 Hz, 1H), 8.06 (dd, J=8.7, 1.9 Hz, 1H), 7.73(d, J=8.7 Hz, 1H), 7.67 (s, 1H), 7.19 (s, 2H), 3.85-3.81 (m, 2H), 3.80(t, J=5.9 Hz, 2H), 3.49 (t, J=5.9 Hz, 2H), 3.20-3.18 (m, 2H), 2.21-2.18(m, 2H), 2.07-2.04 (m, 2H). ¹³C NMR (CD₃OD): δ 184.2, 170.5, 158.7,155.2, 146.8, 141.0, 131.0, 128.52, 128.5, 128.4, 124.5, 116.6, 113.2,110.6, 56.1, 55.7, 37.6, 24.0. LCMS (m/z): 410.3 (100%).

The results of the experiments are now described.

Group II introns, are large self-splicing ribozymes that are found inthe mitochondrial genomes of plants, fungi, and yeast, but are notpresent in mammals. These autocatalytic RNA molecules adopt an elaboratetertiary structure that has been crystallographically characterized andwhich contains an active-site for RNA cleavage and ligation as well assolvent-accessible pockets for potential inhibitor binding (Toor et al.,2008, Science, 320:77-82; Toor et al., 2010, RNA, 16:57-69) (FIG. 22A).In yeasts such as Saccharomyces cerevisiae (S. cerevisiae) and thepathogen Candida parapsilosis (C. parapsilosis), group II introns arefound within genes that are essential for respiration, such ascytochrome oxidase subunit genes of the mitochondria. Importantly,respiration is essential for pathogenic yeast to differentiate intobiofilms, which colonize medical implant surfaces, are relativelyresistant to antifungals, and contribute to pathogenic virulence(Morales et al., 2013, MBio, 4:e00526-00512; Richard et al., 2005,Eukaryot Cell, 4:1493-1502). Thus, based on the complexity of theirstructures, and their essential role in fungal metabolism, group IIintrons represent outstanding targets for the development of highlyspecific antifungal agents.

To identify group II intron splicing inhibitors, a high-throughputscreening-compatible fluorescent assay was developed for monitoringribozyme activity of the well-characterized ai5γ group II intron from S.cerevisiae (FIG. 23A, FIGS. 1A-1D, FIG. 22B). Using this assay, acurated library of 10,000 compounds was screened, identifying 16reproducible hits. Interestingly, a third of these hits contained agallate moiety within the molecular structure (FIG. 1D), suggesting thatthis functionality might contribute to inhibitory activity. A series ofcommercially available analogs of the major hits was analyzed in orderto identify more suitable scaffolds for further optimization. The mostpotent scaffold identified during this phase of the study was compound1, which exhibited an IC₅₀ of 2 μM, (CAS 3260-50-2, FIG. 24 , Table 2).It was used as a starting point for the design of additional compoundsthat were used to define structure-activity relationships (SAR) and tooptimize potency (Table 2).

In Vitro SAR and Optimization of Potency

The primary fluorimetric assay was complemented by a robust secondaryradioanalytic self-splicing assay of the precursor RNA containing thefull length ai5γ intron and short exons, which enabled the determinationof K_(i) values for all compounds of interest (FIG. 22C, FIGS. 25A-Table2).

As part of an initial strategy to optimize the early leads, a series ofcompounds was selected to determine the critical structural componentsrequired for activity: the pharmacophore. Three regions of CAS 3260-50-2(A, B and C) were defined and substituents in these sections of themolecule were evaluated for their effect on inhibition (FIG. 24 ). Inregion A, replacing any of the hydroxyl groups with hydrogen or methoxyor boronic acid substituents (compounds APY-007, APY-014, APY-001,APY-098; Table 2.) all resulted in nearly complete loss of activity.Notably, when the trihydroxyl was replaced with a dihydroxyl catecholmoiety, the molecule was inactivated both in vitro and in vivo (APY-007,Table 2), indicating that presence of the catechol motif by itselfcannot explain reactivity of the group II intron inhibitors, unlikecertain classes of promiscuous molecules that are collectivelyclassified as “PAINS” compounds.

In contrast, each of the hydroxyl groups in region C could be removed orreplaced with halogen atoms without significant loss of function(compounds APY-019, APY-024, APY-083, FIG. 24 , Table 2). To minimizeany risk of reactivity from the α,β-unsaturated ketone, the2-benzylidenebenzofuran-3(2H)-one moiety was replaced with a morechemically and metabolically stable benzofuran-2-yl(phenyl)methanone(region B), This more drug-like molecular template resulted in a 2-foldincrease in in vitro potency (APY-068, FIG. 24 , Table Other attempts tointroduce changes in this region (for example, amide or thiazolederivatives) resulted in a substantial loss of activity (compoundsAPY-077 and APY-093, Table 2). The fact that potency was sensitive tocertain modifications in Region B, and in distal parts of Region C(Table 2), indicates that inhibitory activity is not solely attributableto functional groups in Region A.

With the new lead structure (APY-068) in hand, additional substituentswere added to the benzofuran moiety to further develop thestructure-activity relationship for the series. Introduction of a widevariety of substituents, including aryl, heteroaryl, amino or halogensat the 5′ or 6′-positions were all generally tolerated (compoundsAPY-083, APY-081, APY-084, APY-090, APY-091, APY-094, APY-097,Intronistat A, and Intronistat B, FIG. 24 , Table 2). Notably, the datasuggest that adding large substituents or positively charged residues toregion C can increase inhibitory activity (lower the K_(i)) of therespective compounds (FIG. 24 , Table 2), Examples of such effects areobserved for compounds APY-084, APY-090 and NED-2020 (FIG. 24 , Table2).

Reversibility of inhibitor binding to the intron was established usingpulse-chase dilution experiments (FIG. 25D). The clear reactivitypatterns evident from this SAR analysis, together with facilereversibility of compound binding and inhibition are consistent withselective behavior.

TABLE 2 Structure-activity relationships of representative Group IIintron inhibitors. In vitro data (IC₅₀ and K_(i) values) are highlightedin bold, and MICs for C. parapsilosis are highlighted in italic. Rowsshowing positive correlation between in vitro data and MICs arehighlighted in bold. Rows showing negative correlation between in vitrodata and MIC are shown in regular non-bold. A few cases ofanticorrelated data were observed, but only for highly hydrophobicmolecules expected to participate in pleiotropic interactions in thecell. Compound IC₅₀ K_(i) MIC C. IC₅₀ name ai5γ ai5γ parapsi- HEK- orCAS intron, intron, losis, 293, number Structure MW μM μM μg/ml μg/mlAPY- 001

314.29 >100¹     >1000 >128 >128 Compound 1 (CAS 3260- 50-2)

302.24 2.3 ± 0.4 2.1 ± 0.6 16 34 ± 5  APY- 007

286.24 >100¹ 156 ± 34 >128 >128 APY- 014

286.24 266 ± 37  251 ± 31 >128 >64 APY- 019

286.24 8 ± 2 4.4 ± 0.4 16 >32 APY- 024

270.24 7 ± 1 5.8 ± 0.2 8 23 ± 2  APY- 068

270.24 3.7 ± 0.6 1.94 ± 0.02 16 1.3 ± 0.3 APY- 077

319.31 30 ± 4  44 ± 11 64 >32 APY- 081

349.13 6.8 ± 1.1 5.3 ± 0.2 4-8 >128 APY- 083

339.13 3.3 ± 0.3 0.9 ± 0.1 8 0.5 ± 0.1 APY- 084

376.36 0.9 ± 0.1 1.3 ± 0.4 2-4 1.1 ± 0.1 APY- 090

512.39 2.1 ± 0.4 0.37 ± 0.04 4-8 1.7 ± 0.6 APY- 091

445.46 2.4 ± 0.4 4.5 ± 0.9 8 5.35 ± 0.05 APY- 093

496.6 86 ± 3  53 ± 5  32-64 7.2 ± 1.5 APY- 094

481.52 9.9 ± 0.7 3.39 ± 0.01  8-16 15 ± 4  APY- 097

415.2 2.8 ± 0.2 3.9 ± 0.5 8 5.4 ± 0.6 APY- 098

390.17 >1000      >1000 >128 11 ± 3  APY- 101 (Introni- stat B)

388.37 1.5 ± 0.1 2.1 ± 0.2 2-4 >128 NED- 2020 (Introni- stat B)

410.43 3.8 ± 0.5 0.36 ± 0.02 2-4 >64 “Com- pound 2”

318.28 7.3 8 32 34 APY- 003

344.32 >500      >128 >64 APY- 006

298.06 not determined 55 APY- 010

306.22 not determined 140 >128 0.55 APY- 012

314.29 >500      >128 APY- 013

286.24 16.7  16.5 32 19 APY- 018

314.1 not determined 660 APY- 021

288.26 14   9.4 32 APY- 022

304.26 5.5 6.5 32 17.6 APY- 031

324.24 6.3 32 47.8 APY- 032

288.26 10.6  8 4.14 APY- 033

322.7 6.5 8 50 APY- 034

322.7 4.7 8 54.5 APY- 035

348.31 7.7 4 39.3 APY- 036

348.31 7.7 4 10.4 APY- 037

304.26 3.7 8 5.4 APY- 038

334.28 1.3 1.1 8 15.9 APY- 040

288.26 9.9 8 30.1 APY- 041

332.31 3.8 8 49.5 APY- 054

302.29 >500      >128 >64 APY- 056

fw = 440.24 2.7 ± 0.5 1.7 32 1.1 APY- 057

274.24 12.9  16 39.4 APY- 058

fw = 362.87 10.1  4 23 APY- 069

348.35 27   29 32 >128 APY- 070

288.23 4.2 1 32 >128 APY- 071

320.3 0.3 0.15 16 6 APY- 076

308.33 8   5.4 16 7 APY- 078

333.34 27   16 64 >128 APY- 079

323.34 20   32 32 4.6 APY- 085

285.29 6.4 32 18.7 APY- 086

299.32 61   64 43.7 APY- 087

313.35 79   32 8.4 APY- 088

372.35 24   16 6.55 APY- 092

446.56 1.3 16 24.8 APY- 095

491.92 inconclusive (fluorescent molecule) 4-8 2.5 APY- 096

507.53 inconclusive (fluorescent molecule) 2-4 1.4 APY- 099

406.45 >1000      >128 8.2 APY- 100

373.36 >1000      >128 >128 NED- 2014

319.76 46 ± 2  not determined 8 0.6 NED- 2015

314.34 >1000      not determined 16-32 4.4 NED- 2016

327.28 >1000      not determined 4 1.5 NED- 2017

314.25 >1000      not determined 16-32 NED- 2018

384.39 9 ± 4 0.62 ± 0.03 16 >128 NED- 2019

412.44 4.8 ± 0.5 0.49 ± 0.03 8 >64 NED- 2021

417.42 >1000      not determined 32-64 NED- 2022

424.45 52 ± 12 not determined 16 NED- 2023

425.45 6 ± 2 not determined 16 36 NED- 2034

280.34 >1000      not determined  8-16 22 NED- 2035

398.24 55 ± 4  not determined >128 1.6 NED- 2036

332.31 14 ± 1  not determined 8 8.2 NED- 2037

338.32 1.8 ± 0.5 not determined 16 2.8 NED- 2038

417.22 2.7 ± 0.5 1.7 32 1.1 NED- 2039

362.34 6 ± 1 not determined 8 NED- 2040

493.53 >1000      not determined 8 7.5 NED- 2041

415.48 12 ± 4  not determined 32 NED- 2042

427.49 >1000      not determined 16 NED- 2047

184.15 77 ± 3  not determined 16-32 NED- 2048

315.33 4 ± 1 not determined 4-8 9.8 NED- 2134

322.32 3.3 ± 1  not determined 2-4 4.2 NED- 2135

332.31 0.8 ± 0.2 not determined 2 2.4 NED- 2136

332.31 1.35 ± 0.15 not determined 2 1 NED- 2173

352.35 6 ± 1 not determined 8 8.6 NED- 2174

376.37 5 ± 1 not determined 4-8 2.7 NED- 2175

429.43 2.3 ± 0.8 not determined 4-8 14 NED- 2251

278.31 not determined not determined 16-32 ¹No activity was detectedbetween 5 nM and 100 μM of the compound. Measurements at concentrationsabove 100 μM were precluded by fluorescence of the compound at highconcentrations.Inhibition of Group II Splicing in S. cerevisiae In Vivo.

It was important to determine whether compounds that disrupt splicing ofthe ai5γ intron in vitro can also inhibit splicing of this intron invivo. The ai5γ group II intron interrupts a gene encoding the firstsubunit of the cytochrome oxidase (COX1) in S. cerevisiae, andrespiratory-deficient petite colonies are therefore formed when ai5γintron splicing is disrupted. These colonies cannot grow onnon-fermentable carbon sources like glycerol, but they exhibit growth onglucose (Perlman, 1990, Methods in enzymology, 181:539-558). Todetermine whether the inhibitory compounds might induce a respiratorydefect, the growth of S. cerevisiae was compared in both YPD and YPGEmedia in the presence of a panel of inhibitors. Growth of S. cerevisiaein the presence of inhibitory small molecules is inhibited inglycerol/ethanol medium (YPGE), but not in glucose medium (YPD) (Table1), which is consistent with the expected respiratory detect.

To analyze whether the growth defect Observed in the presence ofinhibitor is specifically due to defective splicing of the ai5 g intron,whether the compounds inhibited growth of an “intronless strain”, whichcontains an intact COX1 subunit gene from which the intron has beenremoved, thereby obviating the requirement for splicing (Perez-Martinezet al., 2003, EMBO J, 22:5951-5961) was examined. A clear difference wasobserved in growth in between the intronless strain and the wild-typestrain in the presence of tritronistat A, revealing that the intronlessstrain is much more resistant to Intronistat A in YPGE medium (FIGS.26A-26B, FIG. 27A). This behavior is consistent with specific inhibitionof the ai5γ intron in vivo. That said, growth of the intronless strainis somewhat slower in the presence of inhibitor, suggesting thatIntronistat A may cause certain off-target effects.

To directly monitor the effect of the most potent compounds on group IIintron splicing in vivo, a qRT-PCR. assay was developed for monitoringthe splicing of the ai5γ intron in S. cerevisiae in the presence ofsmall molecules. Small molecules that are active in vitro cause a severein vivo splicing defect as evident from significant accumulation ofprecursor RNA molecules containing 5′-exon-intron junction (FIGS.26A-26B and FIGS. 27A-27B). Importantly, unspliced COX1 transcripts aretargeted for rapid degradation in cells (Dziembowski et al., 2003, JBiol Chem, 278:1603-1611), so the direct observation of substantialprecursor accumulation suggests a considerable effect on splicing. Takentogether, these results in cells demonstrate that the highest-affinitycompounds specifically target the ai5γ intron in vivo, selectivelydisrupting splicing of the COM gene and thereby reducing yeast growth.

Selectivity of the Group II Intron Inhibitors.

Although the SAR, reversibility analysis and gene specificity of thecompounds are consistent with selectivity, it was important to determinewhether the compounds can also bind other highly structured RNAmolecules, and other RNA splicing systems. In addition, it wasquestioned whether activity requires the fully-folded group II intronRNA tertiary structure, or if individual intron domains can bind theinhibitors with high affinity. To this end, the inhibitory activity ofone of the most promising compounds, Intronistat B, was monitored in thepresence of a large excess of various RNAs, including separate ai5γintron domains D1, D3, D56, the U2-U6 snRNA stemloop (analogous to groupII intron D5), and yeast tRNA_(Phe) (FIGS. 23A-23B). The latter was animportant control because tRNA molecules possess many archetypalelements of RNA tertiary structure, such as kissing loops involvingcanonical and non-canonical base pairs, coaxially stacked helices, basetriples and U-turn motifs, which make them commonly used specificitycontrols for RNA targeting (Luedtke et al., 2003, Biochemistry,42:11391-11403). However, none of these RNAs, presented in a 1000-foldexcess relative to intron RNA (2 nM), affect the inhibitory activity ofIntronistat B (FIG. 28A). The only RNA that competed with radiolabeledSE group II intron RNA for binding of Intronistat B was the sameunlabeled group IIB intron RNA added in excess (FIG. 28A). To evaluateinhibition of the other two known RNA splicing systems (group I andspliceosome) splicing of the Azoarcus pre-tRNA (Ile) group I intron wasmonitored (Tanner et al., 1996, RNA 2:74-83) in the presence ofIntronistat B. Splicing of the Azoarcus intron is unaffected, even at100 μM Intronistat B (FIG. 28B). In addition, qRT-PCR was used tomonitor inhibition of group I intron and spliceosomal splicing in S.cerevisiae in vivo. Consistent with previous results, the inhibitorsonly affect splicing of the yeast ai5γ group II intron (FIG. 26B).Spliceosomal processing, group I intron splicing and even splicing ofgroup II introns from subclasses that differ from subclass IIB areunaffected by the small molecule inhibitors. These results areconsistent with the in vitro results and suggest that the inhibitorsbind selectively to group IIB introns. The data also indicate that theinhibitors bind tertiary structural elements formed by the entire intronand not individual intronic domains.

Small Molecule Growth Inhibition of C. parapsilosis

The yeast pathogen C. parapsilosis contains a single group IIB intron inits COX1 gene (Li et al., 2011, RNA, 17:1321-1335). The active site ofthis intron (D5) is almost identical to that of the ai5 g intron (FIG.1B), suggesting that compounds that inhibit the S.c. ai5γ intron mayalso inhibit splicing by the group II intron in C. parapsilosis. Toevaluate efficacy of the compounds against this pathogen, the minimuminhibitory concentrations (MIC values) required for growth inhibition ofC. parapsilosis were evaluated. The high affinity ai5γ intron inhibitorssignificantly reduce the growth of C. parapsilosis (FIG. 24 , Table 2).Correlations between IC₅₀, K_(i) and MIC values suggest that thesecompounds employ the same mechanism of action both in vitro and in vivo(FIG. 24 , Table 2). While many of the highest-affinity compoundsdisplayed strong MIC values, compounds Intronistat A (K_(i)=2.1±0.2 μM)and Intronistat B (K_(i)=0.36±0.02 μM) were particularly notable becausetheir MIC values (2-4 μg/ml) are comparable to that of Amphotericin B,which is still commonly used for acute C. parapsilosis infection (Moenet al., 2009, Drugs, 69:361-92) (MIC is 0.5-1 μg/ml) (FIG. 24 , Table2). Given their potency and antifungal effects, compound APY-101 hasbeen named Intronistat A, and compound NED-2020 Intronistat B. Todirectly monitor behavior of the compounds in vivo, qRT-PCR was used toquantify levels of splicing for the C. parapsilosis COX1 precursor mRNA(which contains the group IIB intron) in the presence of Intronistat Band inactive APY-001. A moderate splicing defect caused by Intronistat Bwas identified as indicated by increased levels of unspliced C.p.COX1relative to total, and this effect was not observed in the presence ofinactive APY-001 (FIG. 27C). To determine whether inhibition is specificto yeast, the toxicity of the most potent compounds in human cells wasevaluated, determining the IC₅₀ for inhibition of HEK-293T cells (FIG.24 , FIGS. 29A-29B, Table 2). While some of the compounds are broadlytoxic to all eukaryotic cells tested, the most potent compounds,including Intronistat A and Intronistat B, did not show toxicity inhuman cells after 24 hours incubation (FIG. 24 , FIG. 29A, Table 2).Even after 72 hours incubation Intronistat B had only mild effects oncell viability (FIG. 29B), suggesting that this compound specificallytargets yeast strains that contain group II introns in an essential geneand that it lacks cross-reactivity with the nuclear spliceosome, orother targets, in yeast and humans.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A compound of Formula (V), or a salt thereof;

wherein: L is a divalent linking group selected from the groupconsisting of a single bond and ethylene; R²¹, R²², R²³, R²⁴, and R²⁵are each independently selected from the group consisting of H, —C₁-C₆alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, aryl, heteroaryl,cycloalkyl, heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂, —OH, —OR²⁷,—S(═O)R²⁷, —S(═O)₂R²⁷, —NHS(═O)₂R²⁷, —C(═O)R²⁷, —OC(═O)R²⁷, —CO₂R²⁷,—OCO₂R²⁷, —CH(R²⁷)₂, —N(R²⁷)₂, —C(═O)N(R²⁷)₂, —C(═O)NHR²⁷,—OC(═O)N(R²⁷)₂, —NHC(═O)NH(R²⁷), —NHC(═O)R²⁷, —NHC(═O)OR²⁷,—C(OH)(R²⁷)₂, and —C(NH₂)(R²⁷)₂, and combinations thereof; or optionallytwo adjacent R²¹-R²⁵ are joined to form a ring; each occurrence of R²⁷is independently selected from the group consisting of H, —C₁-C₆ alkyl,—C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, aryl, heteroaryl, cycloalkyl,heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂, —OH, and combinationsthereof; and R²⁶ is selected from the group consisting of an aryl groupand a heteroaryl group, wherein the aryl or heteroaryl group may beoptionally substituted.
 2. The compound of claim 1, wherein R²², R²³,and R²⁴ are OH.
 3. The compound of claim 1, wherein R²⁶ is a group ofFormula (VI):

wherein in Formula (VI): * represents the attachment to Formula (V); Z¹,Z², Z³, Z⁴, and Z⁵ are independently CR²⁸ or N; each occurrence of R²⁸is independently selected from the group consisting of H, C₁-C₆ alkyl,F, Cl, Br, I, —CN, —NO₂, —OH, —CF₃, —OR²⁹, —N(R²⁹)₂, —C(═O)R²⁹, C₁-C₆heteroalkyl, aryl-(C₁-C₃)alkyl, cycloalkyl, alkynyl, and combinationsthereof; or optionally two adjacent R²⁸ are joined together to form aring; and R²⁹ is selected from the group consisting of H, C₁-C₆ alkyl,and heteroaryl-(C₁-C₃)alkyl.
 4. The compound of claim 1, wherein thecompound of Formula (V) is selected from the group consisting of:


5. A method of reducing, or preventing growth of an organism harboringan active group II intron, the method comprising contacting the organismwith an effective amount of an inhibitor of group II intron splicing. 6.The method of claim 5, wherein the inhibitor of group II intron splicingis at least one selected from the group consisting of a protein, apeptide, a peptidomimetic, an antibody, a ribozyme, a small moleculechemical compound, a nucleic acid, an aptamer, a modified nucleic acid,a vector, and an antisense nucleic acid molecule.
 7. The method of claim5, wherein the inhibitor of group II intron splicing is at least one ofthe following: a compound of Formula (I), or a salt thereof;

wherein in Formula (I): X is O, S, or NR¹⁰; R¹, R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, and R¹⁰ are each independently selected from the groupconsisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl,aryl, heteroaryl, cycloalkyl, heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂,—OH, —OR¹¹, —SR¹¹, —S(═O)R¹¹, —S(═O)₂R¹¹, —NHS(═O)₂R¹¹, —C(═O)R¹¹,—OC(═O)R¹¹, —CO₂R¹¹, —OCO₂R¹¹, —CH(R¹¹)₂, —N(R¹¹)₂, —C(═O)N(R¹¹)₂,—C(═O)NHR¹¹, —OC(═O)N(R¹¹)₂, —NHC(═O)NH(R¹¹), —NHC(═O)R¹¹, —NHC(═O)OR¹¹,—C(OH)(R¹¹)₂, and —C(NH₂)(R¹¹)₂, and combinations thereof; or optionallytwo adjacent groups within R¹-R⁵ or within R⁶-R⁹ are joined to form aring; each occurrence of R¹¹ is independently selected from the groupconsisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl,aryl, heteroaryl, cycloalkyl, heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂,—OH, and combinations thereof; and wherein only one of the two followingconditions is met: (a) the bond between carbons 1 and 2 is a singlebond, the bond between carbons 2 and 3 is a double bond, Y is (═O), andZ is H; or (b) the bond between carbons 1 and 2 is a double bond, thebond between carbons 2 and 3 is a single bond, Y is H, and Z is (═O); acompound of Formula (V), or a salt thereof;

wherein in Formula (V): L is a divalent linking group selected from thegroup consisting of a single bond and ethylene; R²¹, R²², R²³, R²⁴, andR²⁵ are each independently selected from the group consisting of H,—C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, aryl, heteroaryl,cycloalkyl, heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂, —OH, —SR²⁷,—S(═O)R²⁷, —S(═O)₂R²⁷, —NHS(═O)₂R²⁷, —C(═O)R²⁷, —OC(═O)R²⁷, —CO₂R²⁷,—OCO₂R²⁷, —CH(R²⁷)₂, —N(R²⁷)₂, —C(═O)N(R²⁷)₂, —C(═O)NHR²⁷,—OC(═O)N(R²⁷)₂, —NHC(═O)NH(R²⁷), —NHC(═O)R²⁷, —NHC(═O)OR²⁷,—C(OH)(R²⁷)₂, and —C(NH₂)(R²⁷)₂, and combinations thereof; or optionallytwo adjacent R²¹-R²⁵ are joined to form a ring; each occurrence of R²⁷is independently selected from the group consisting of H, —C₁-C₆ alkyl,—C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, aryl, heteroaryl, cycloalkyl,heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂, —OH, and combinationsthereof; and R²⁶ is selected from the group consisting of an aryl groupand a heteroaryl group, wherein the aryl or heteroaryl group may beoptionally substituted.
 8. A method of treating or ameliorating adisease associated with an organism harboring an active group II intronin a subject, the method comprising administering an effective amount ofa composition comprising an inhibitor of group II intron splicing to thesubject.
 9. The method of claim 8, wherein the disease or disorder isselected from the group consisting of a bacterial infection, a yeastinfection, a fungal infection, and a parasite infection.
 10. The methodof claim 8, wherein the subject is a mammal.
 11. The method of claim 8,wherein the mammal is a human.
 12. The method of claim 8, wherein theinhibitor of group II intron splicing is at least one selected from thegroup consisting of a protein, a peptide, a peptidomimetic, an antibody,a ribozyme, a small molecule chemical compound, a nucleic acid, anaptamer, a modified nucleic acid, a vector, and an antisense nucleicacid molecule.
 13. The method of claim 8, wherein the inhibitor of groupII intron splicing is at least one of the following: a compound ofFormula (I), or a salt thereof;

wherein in Formula (I): X is O, S, or NR¹⁰; R¹, R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, and R¹⁰ are each independently selected from the groupconsisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl,aryl, heteroaryl, cycloalkyl, heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂,—OH, —OR¹¹, —SR¹¹, —S(═O)R¹¹, —S(═O)₂R¹¹, —NHS(═O)₂R¹¹, —C(═O)R¹¹,—OC(═O)R¹¹, —CO₂R¹¹, —OCO₂R¹¹, —CH(R¹¹)₂, —N(R¹¹)₂, —C(═O)N(R¹¹)₂,—C(═O)NHR¹¹, —OC(═O)N(R¹¹)₂, —NHC(═O)NH(R¹¹), —NHC(═O)R¹¹, —NHC(═O)OR¹¹,—C(OH)(R¹¹)₂, and —C(NH₂)(R¹¹)₂, and combinations thereof; or optionallytwo adjacent groups within R¹-R⁵ or within R⁶-R⁹ are joined to form aring; each occurrence of R¹¹ is independently selected from the groupconsisting of H, —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl,aryl, heteroaryl, cycloalkyl, heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂,—OH, and combinations thereof; and wherein only one of the two followingconditions is met: (a) the bond between carbons 1 and 2 is a singlebond, the bond between carbons 2 and 3 is a double bond, Y is (═O), andZ is H; or (b) the bond between carbons 1 and 2 is a double bond, thebond between carbons 2 and 3 is a single bond, Y is H, and Z is (═O); acompound of Formula (V), or a salt thereof;

wherein in Formula (V): L is a divalent linking group selected from thegroup consisting of a single bond and ethylene; R²¹, R²², R²³, R²⁴, andR²⁵ are each independently selected from the group consisting of H,—C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, aryl, heteroaryl,cycloalkyl, heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂, —OH, —SR²⁷,—S(═O)R²⁷, —S(═O)₂R²⁷, —NHS(═O)₂R²⁷, —C(═O)R²⁷, —OC(═O)R²⁷, —CO₂R²⁷,—OCO₂R²⁷, —CH(R²⁷)₂, —N(R²⁷)₂, —C(═O)N(R²⁷)₂, —C(═O)NHR²⁷,—OC(═O)N(R²⁷)₂, —NHC(═O)NH(R²⁷), —NHC(═O)R²⁷, —NHC(═O)OR²⁷,—C(OH)(R²⁷)₂, and —C(NH₂)(R²⁷)₂, and combinations thereof; or optionallytwo adjacent R²¹-R²⁵ are joined to form a ring; each occurrence of R²⁷is independently selected from the group consisting of H, —C₁-C₆ alkyl,—C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, aryl, heteroaryl, cycloalkyl,heterocycloalkyl, F, Cl, Br, I, —CN, —NO₂, —OH, and combinationsthereof; and R²⁶ is selected from the group consisting of an aryl groupand a heteroaryl group, wherein the aryl or heteroaryl group may beoptionally substituted.
 14. The method of claim 8, wherein the organismis selected from the group consisting of a bacteria, a yeast, a fungus,a protist, a parasite, and a plant.
 15. The method of claim 8, whereinadministration of the inhibitor reduces or prevents in the organism atleast one of biofilm formation and algae formation.